EP0965033A1 - Detection directe de biocatalyseurs par procede colorimetrique - Google Patents

Detection directe de biocatalyseurs par procede colorimetrique

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Publication number
EP0965033A1
EP0965033A1 EP98907684A EP98907684A EP0965033A1 EP 0965033 A1 EP0965033 A1 EP 0965033A1 EP 98907684 A EP98907684 A EP 98907684A EP 98907684 A EP98907684 A EP 98907684A EP 0965033 A1 EP0965033 A1 EP 0965033A1
Authority
EP
European Patent Office
Prior art keywords
sep
pda
biopolymeric
liposomes
materials
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98907684A
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German (de)
English (en)
Inventor
Deborah Charych
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP0965033A1 publication Critical patent/EP0965033A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G01N2405/06—Glycophospholipids, e.g. phosphatidyl inositol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/08—Sphingolipids
    • G01N2405/10—Glycosphingolipids, e.g. cerebrosides, gangliosides

Definitions

  • phospholipase D upases in general (e.g., triacylglycerol lipases, lipoprotein lipases, and pancreatic lipases), other membrane modifying enzymes (e.g., lipolytic enzymes, acyltransferases, protein kinases, and glycosidase), and any other natural or artificial membrane modifying events.
  • upases e.g., triacylglycerol lipases, lipoprotein lipases, and pancreatic lipases
  • other membrane modifying enzymes e.g., lipolytic enzymes, acyltransferases, protein kinases, and glycosidase
  • any other natural or artificial membrane modifying events e.g., lipolytic enzymes, acyltransferases, protein kinases, and glycosidase
  • methods and compositions that provide simple detection of the modifying events and that allow high throughput screening of inhibitors are desired.
  • the present invention relates to methods and compositions for the direct detection of membrane conformational changes through the detection of color changes in biopolymeric materials.
  • the present invention allows for the direct colorimetric detection of membrane modifying reactions and analytes responsible for such modifications and for the screening of reaction inhibitors.
  • the presently claimed invention provides methods for detecting a reaction, comprising: providing biopolymeric material comprising reaction substrate and a plurality of self- assembling monomers, and a reaction means; exposing the reaction means to the biopolymeric material; and detecting a color change in the biopolymeric material which indicates at least a partial occurrence of the reaction.
  • the method further comprises the step of quantifying the color change in the biopolymeric material.
  • the reaction means comprises a lipid cleavage means.
  • the cleavage means comprises a lipase.
  • the lipase is selected from the group consisting of phospholipase A 2 , phospholipase C, and phospholipase D.
  • the biopolymeric materials are selected from the group consisting of liposomes, films, tubules, helical assemblies, fiberlike assemblies, and solvated polymers.
  • the self assembling monomers of the biopolymeric materials comprise diacetylene monomers.
  • the self assembling monomers comprise diacetylene monomers selected from the group consisting of 5,7-docosadiynoic acid, 5,7-pentacosadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof.
  • the self-assembling monomers are selected from the group consisting of acetylenes, alkenes, thiophenes, polythiophenes, siloxanes, poly- silanes, anilines, pyrroles, polyacetylenes, poly (para-phylenevinylene), poly (para-phylene), vinylpyridinium, and combinations thereof.
  • the presently claimed invention provides methods wherein the biopolymeric material further comprises one or more ligands.
  • the ligand is selected from the group consisting of proteins, antibodies, carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, short peptides, pepstatin.
  • the ligands have affinity for the reaction means.
  • the presently claimed invention also provides methods wherein the biopolymeric material further comprises one or more dopants.
  • the dopant is selected from the group consisting of surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, steroids, cerebroside, lysophosphatidylcholine, D- erythroshingosine.
  • surfactants polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine,
  • the dopants comprise diacetylene derivatives selected from the group consisting of sialic acid-derived diacetylene, lactose-derived diacetylene, amino acid-derived diacetylene, and combinations thereof.
  • the biopolymeric material further comprises a support, wherein the biopolymeric material is immobilized to the support.
  • the support is selected from the group consisting of polystyrene, polyethylene, teflon, mica, sephadex, sepharose, polyacrynitriles, filters, glass, gold, silicon chips, and silica.
  • the presently claimed invention further provides methods for detecting the presence of an analyte, comprising providing biopolymeric material comprising analyte substrate and a plurality of self-assembling monomers; exposing a sample suspected of containing the analyte to the biopolymeric material; and detecting a color change in the biopolymeric material, which indicates the presence of the analyte.
  • the analyte comprises a lipid cleavage means.
  • the cleavage means comprises a lipase.
  • the lipase is selected from the group consisting of phospholipase A 2 , phospholipase C, and phospholipase D.
  • the biopolymeric material further comprises one or more ligands. In certain embodiments, the ligands have affinity for the analyte.
  • the presently claimed invention further provides methods for detecting inhibitors, comprising: providing biopolymeric material comprising reaction substrate and a plurality of self-assembling monomers, a reaction means, and a sample suspected of containing an inhibitor; combining the biopolymeric material and the sample suspected of containing an inhibitor; exposing the biopolymeric material and the sample suspected of containing an inhibitor to the reaction means; and detecting a color change in the biopolymeric material, thereby detecting the activity of the inhibitor.
  • the detecting a color change in the biopolymeric material comprises comparing the color change to one or more control samples.
  • the method further comprises the step of quantitating the color change in the biopolymeric material.
  • the reaction means comprises a lipid cleavage means.
  • the cleavage means comprises a lipase.
  • the lipase is selected from the group consisting of phospholipase A 2 , phospholipase C, and phospholipase D.
  • Figure 1 shows a schematic representation of biopolymeric films.
  • Y is a centrosymmetric multilayer film, while films X and Z are noncentrosymmetric multilayers.
  • Figure 2 shows a schematic representation of biopolymeric liposomes.
  • Part A is a cross-section two-dimensional view and part B is a three-dimensional view of half of a liposome.
  • Figure 3 shows biopolymeric 1) liposomes and 2) films comprising the same biopolymeric material and exposed to the same analyte.
  • Figure 4 shows a heating curve depicting the large main phase transition for unpolymerized liposomes prepared from PDA monomer.
  • Figure 5 shows a schematic representation of a Langmuir Blodgett apparatus where a compressed film is being transferred to a vertical plate.
  • Figure 6 shows a micrograph of liposomes cooled only to room temperature.
  • Figure 7 shows a micrograph of liposomes prepared with cooling to 4°C.
  • Figure 8 shows the chemical structure of 5,7-pentacosadiynoic acid.
  • Figure 9 shows a synthesis reaction for modifying the free amino group of a molecule for coupling to a lipid monomer.
  • Figure 10 shows the properties of biopolymeric materials composed of amino acid- derivated diacetylene monomers.
  • Figure 1 1 shows the chemical structure of sialic acid derived 10,12-pentacosadiynoic acid (compound 1) and 10,12-pentacosadiynoic acid (compound 2).
  • Figure 12 shows substrate lipid (i.e., DMPC) in a diacetylenic lipid matrix before (top) and after (bottom) polymerization.
  • substrate lipid i.e., DMPC
  • Figure 13 shows the visible absorption spectrum of the liposomes of Figure 12 before (solid line) and after (dashed line) exposure to phospholipase A 2 .
  • Figure 14 shows the change in colorimetric response of the liposomes of Figure 12 with varying concentrations of DMPC in response to phospholipase A 2 exposure.
  • Figure 15 shows the absorbance at 412 nm of liposomes containing l,2-bis-(S- decanoyl)-l,2-dithio-sn-glycero-3-phosphocholine (DTPC) following exposure to PLA 2 for various lengths of time.
  • Figure 16 shows 31 P NMR spectra of the DMPC/PDA vesicles prior to the addition of PLA 2 (A), and following the enzymatic reaction (B).
  • Figure 17 shows the colorimetric response of DMPC containing liposomes in the presence of PLA 2 (circles), and PLA 2 with inhibitors (squares and diamonds).
  • Figure 18 shows the visible absorption spectra of the polydiacetylene liposomes in a sol-gel matrix.
  • Figure 19 shows the visible absorption spectra of the material in Figure 18 following heating of the liposomes to 55 °C.
  • Figure 20 shows an optical micrograph of diacetylene film.
  • Figure 21 shows the properties of polydiacetylene monolayers with and without sialic acid-derivated PDA and ganglioside G M1 .
  • Figure 22 shows the isotherms of 5% G M ,/5% SA-PDA 90% PDA as a function of subphase concentration of CdCl 2 .
  • Figure 23 shows the isotherms of 5% G M1 /5% SA-PDA/90% PDA at pH 4.5, 5.8, and 9.2.
  • Figure 24 shows the temperature effect on the isotherms of 100% PDA, 5%SA- PDA/95% PDA, and 5% G M1 /5% SA-PDA/90% PDA.
  • Figure 25 shows the visible abso ⁇ tion spectrum of "blue phase” 5% G M1 and 95% 5,7-docosadiynoic acid liposomes.
  • Figure 26 shows the visible absorption spectrum of the liposomes of Figure 25 following exposure to cholera toxin.
  • Figure 27 shows the visible absorption spectrum for sialic-acid containing films before
  • Figure 28 shows the color transition of ganglioside G M1 -containing liposomes in response to varying concentrations of cholera toxin.
  • Figure 29 shows the visible abso ⁇ tion spectrum of the polymeric liposomes containing 5% G M1 ligand and 95% 5,7-DCDA.
  • Figure 30 shows the visible abso ⁇ tion spectrum of the material in Figure 29 following exposure to E coli toxin.
  • Figure 31 shows the abso ⁇ tion spectrum of a PCA film in before (line a) and after exposure to 1-octanol dissolved in water (line b).
  • Figure 32 shows a bar graph indicating colorimetric responses of PDA material to various VOCs (A) and a table showing the concentration of the VOCs (B).
  • Figure 33 shows a graph comparing colorimetric responses of biopolymeric material to 1-butanol to the concentration of 1-butanol.
  • Figure 34 shows compounds and synthesis schematics for producing PDA derivatives for the detection of small organic compounds.
  • Figure 36 shows the colorimetric response of hexokinase containing biopolymeric material to a variety of sugars.
  • Figure 37 shows derivations of PDA for use in detection arrays.
  • Figure 38 shows the organic synthesis of compound 2.10 from Figure 37.
  • reaction refers to any change or transformation in which a substance (e.g., molecules, membranes, and molecular assemblies) combines with other substances, interchanges constituents with other substances, decomposes, rearranges, or is otherwise chemically altered.
  • reaction means refers to any means of initiating and/or catalyzing a reaction. Such reaction means include, but are not limited to, enzymes, temperature changes, and pH changes.
  • affinity for said reaction means refers to compounds with the ability to specifically associate (e.g., bind) to a given reaction mean, although not necessarily a substrate for the reaction means.
  • a PLA 2 antibody has affinity for PLA 2 , but is not the substrate for the enzyme.
  • immobilization refers to the attachment or entrapment, either chemically or otherwise, of material to another entity (e.g., a solid support) in a manner that restricts the movement of the material.
  • biopolymeric material refers to materials composed of polymerized biological molecules (e.g., lipids, proteins, carbohydrates, and combinations thereof). Such materials include, but are not limited to, films, vesicles, liposomes, multilayers, aggregates, membranes, and solvated polymers (e.g., polythiophene aggregates such as rods and coils in solvent). Biopolymeric material can contain molecules that are not part of the polymerized matrix (i.e., molecules that are not polymerized).
  • protein is used in its broadest sense to refer to all molecules or molecular assemblies containing two or more amino acids. Such molecules include, but are not limited to, proteins, peptides, enzymes, antibodies, receptors, lipoproteins, and glycoproteins.
  • antibody refers to a glycoprotein evoked in an animal by an immunogen (antigen).
  • An antibody demonstrates specificity to the immunogen, or, more specifically, to one or more epitopes contained in the immimogen.
  • Native antibody comprises at least two light polypeptide chains and at least two heavy polypeptide chains. Each of the heavy and light polypeptide chains contains at the amino terminal portion of the polypeptide chain a variable region (i.e., VH and VL respectively), which contains a binding domain that interacts with antigen.
  • Each of the heavy and light polypeptide chains also comprises a constant region of the polypeptide chains (generally the carboxy terminal portion) which may mediate the binding of the immunoglobulin to host tissues or factors influencing various cells of the immune system, some phagocytic cells and the first component (Clq) of the classical complement system.
  • the constant region of the light chains is referred to as the "CL region,” and the constant region of the heavy chain is referred to as the "CH region.”
  • the constant region of the heavy chain comprises a CHI region, a CH2 region, and a CH3 region. A portion of the heavy chain between the CHI and CH2 regions is referred to as the hinge region (i.e., the "H region").
  • the constant region of the heavy chain of the cell surface form of an antibody further comprises a spacer-transmembranal region (Ml) and a cytoplasmic region (M2) of the membrane carboxy terminus.
  • the secreted form of an antibody generally lacks the Ml and M2 regions.
  • biopolymeric films refers to polymerized organic films that are used in a thin section or in a layer form. Such films can include, but are not limited to, monolayers and bilayers. Biopolymeric films can mimic biological cell membranes (e.g., in their ability to interact with other molecules such as proteins or analytes).
  • sol-gel refers to preparations composed of porous metal oxide glass structures. Such structures can have biological or other material entrapped within the porous structures.
  • sol-gel matrices refers to the structures comprising the porous metal oxide glass with or without entrapped material.
  • sol-gel material refers to any material prepared by the sol-gel process including the glass material itself and any entrapped material within the porous structure of the glass.
  • sol-gel method refers to any method that results in the production of porous metal oxide glass. In some embodiments, “sol-gel method” refers to such methods conducted under mild temperature conditions.
  • sol-gel glass and “metal oxide glass” refer to glass material prepared by the sol-gel method and include inorganic material or mixed organic/inorganic material.
  • the materials used to produce the glass can include, but are not limited to, aluminates, aluminosilicates, titanates, ormosils (organically modified silanes), and other metal oxides.
  • direct colorimetric detection refers to the detection of color changes without the aid of an intervening processing step (e.g., conversion of a color change into an electronic signal that is processed by an inte ⁇ reting device). It is intended that the term encompass visual observing (e.g., observing with the human eye) as well as detection by simple spectrometry.
  • the term “analytes” refers to any material that is to be analyzed. Such materials can include, but are not limited to, ions, molecules, antigens, bacteria, compounds, viruses, cells, antibodies, and cell parts.
  • selective binding refers to the binding of one material to another in a manner dependent upon the presence of a particular molecular structure (i.e., specific binding). For example, a receptor will selectively bind ligands that contain the chemical structures complementary to the ligand binding site(s). This is in contrast to "non- selective binding," whereby interactions are arbitrary and not based on structural compatibilities of the molecules.
  • biosensors refers to any sensor device that is partially or entirely composed of biological molecules.
  • the term refers to "an analytical tool or system consisting of an immobilized biological material (such as enzyme, antibody, whole cell, organelle, or combination thereof) in intimate contact with a suitable transducer device which will convert the biochemical signal into a quantifiable electrical signal” (Gronow, Trends Biochem. Sci. 9: 336 [1984]).
  • the term "transducer device” refers to a device that is capable of converting a non-electrical phenomenon into electrical information, and transmitting the information to a device that inte ⁇ rets the electrical signal.
  • Such devices can include, but are not limited to, devices that use photometry, fluorimetry, and chemiluminescence; fiber optics and direct optical sensing (e.g., grating coupler); surface plasmon resonance; potentiometric and amperometric electrodes; field effect transistors; piezoelectric sensing; and surface acoustic wave.
  • miniaturization refers to a reduction in size, such as the size of a sample to increase utility (e.g., portability, ease of handling, and ease of inco ⁇ oration into arrays).
  • the term “stability” refers to the ability of a material to withstand deterioration or displacement and to provide reliability and dependability.
  • the term “conformational change” refers to the alteration of the molecular structure of a substance. It is intended that the term encompass the alteration of the structure of a single molecule or molecular aggregate (e.g., the change in structure of polydiacetylene upon interaction with an analyte).
  • small molecules refers to any molecule with low molecular weight (i.e., less than 10,000 atomic mass units and preferably less than 5,000 atomic mass units) that binds to ligands, interacts with ligands, or interacts with biopolymeric material in a manner that creates a conformational change.
  • pathogen refers to disease causing organisms, microorganisms, or agents including, but not limited to, viruses, bacteria, parasites (including, but not limited to, organisms within the phyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala. and Arthropoda), fungi, and prions.
  • bacteria and "bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia. Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. "Gram negative” and "gram positive” refer to staining patterns obtained with the Gram- staining process which is well known in the art (See e.g., Finegold and Martin, Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15).
  • membrane refers to, in its broadest sense, a sheet or layer of material. It is intended that the term encompass all "biomembranes” (i.e., any organic membrane including, but not limited to, plasma membranes, nuclear membranes, organelle membranes, and synthetic membranes). Typically, membranes are composed of lipids, proteins, glycolipids. steroids, sterols and/or other components. As used herein, the term “membrane fragment” refers to any portion or piece of a membrane. The term “polymerized membrane” refers to membranes that have undergone partial or complete polymerization.
  • membrane rearrangement and “membrane conformational change” refer to any alteration in the structure of a membrane. Such alterations can be caused by physical perturbation, heating, enzymatic and chemical reactions, among other events. Reactions that can result in membrane rearrangement include, but are not limited to lipid cleavage, polymerization, lipid flipping, transmembrane signalling, vesicle formation, lipidation, glycosylation, ion channeling, molecular rearrangement, and phosphorylation.
  • Enzymatic catalysis that results in membrane rearrangement can result from free enzymes interacting with the biopolymeric material (e.g., reacting with an enzyme substrate in the biopolymeric material) and can result from enzymatic activity present in certain analytes (e.g., viruses, bacteria, and toxins among others).
  • lipid cleavage refers to any reaction that results in the division of a lipid or lipid-comprising material into two or more portions.
  • “Lipid cleavage means” refers to any means of initiating and/or catalyzing lipid cleavage. Such lipid cleavage means include, but are not limited to enzymes, free radical reactions, and temperature changes.
  • polymerization encompasses any process that results in the conversion of small molecular monomers into larger molecules consisting of repeated units. Typically, polymerization involves chemical crosslinking of monomers to one another.
  • membrane receptors refers to constituents of membranes that are capable of interacting with other molecules or materials. Such constituents can include, but are not limited to, proteins, lipids, carbohydrates, and combinations thereof.
  • volatile organic compound or “VOC” refers to organic compounds that are reactive (i.e., evaporate quickly, explosive, corrosive, etc.), and typically are hazardous to human health or the environment above certain concentrations.
  • VOCs include, but are not limited to, alcohols, benzenes, toluenes, chloroforms, and cyclohexanes.
  • enzyme refers to molecules or molecule aggregates that are responsible for catalyzing chemical and biological reactions. Such molecules are typically proteins, but can also comprise short peptides, RNAs, ribozymes, antibodies, and other molecules.
  • reaction substrate refers to the substrate for a reaction means (e.g., a "substrate lipid” reacted by a lipid cleavage means).
  • analyte substrate refers to a material or substance upon which an analyte reacts.
  • the analyte can be an enzyme and the analyte substrate is an enzyme substrate.
  • the analyte can be a pathogen and the analyte substrate comprises a material or sample that is altered by a "reaction means" associated with the pathogen.
  • lipase refers to any of a group of hydrolytic enzymes that acts on ester bonds in lipids.
  • lipases include, but are not limited to, pancreatic lipase that catalyses the hydrolysis of triacylglycerols, lipoprotein lipase that catalyzes the hydrolysis of triacylglycerols to glycerol and free fatty acids, and phospholipases, among others.
  • phospholipase refers to enzymes that cleave phospholipids by the hydrolysis of carbon- oxygen or phosphorus-oxygen bonds.
  • Phospholipases include, but are not limited to, phospholipases A,, A 2 , C, and D.
  • drug refers to a substance or substances that are used to diagnose, treat, or prevent diseases or conditions. Drugs act by altering the physiology of a living organism, tissue, cell, or in vitro system that they are exposed to. It is intended that the term encompass antimicrobials, including, but not limited to, antibacterial, antifungal, and antiviral compounds. It is also intended that the term encompass antibiotics, including naturally occurring, synthetic, and compounds produced by recombinant DNA technology.
  • peptide refers to any substance composed of two or more amino acids.
  • carbohydrate refers to a class of molecules including, but not limited to, sugars, starches, cellulose, chitin, glycogen, and similar structures.
  • Carbohydrates can also exist as components of glycolipids and glycoproteins.
  • chromophore refers to molecules or molecular groups responsible for the color of a compound, material, or sample.
  • the term "antigen” refers to any molecule or molecular group that is recognized by at least one antibody.
  • an antigen must contain at least one epitope (i.e., the specific biochemical unit capable of being recognized by the antibody).
  • immunogen refers to any molecule, compound, or aggregate that induces the production of antibodies.
  • an immunogen must contain at least one epitope (i.e., the specific biochemical unit capable of causing an immune response).
  • the term “chelating compound” refers to any compound composed of or containing coordinate links that complete a closed ring structure. The compounds can combine with metal ions, attached by coordinate bonds to at least two of the nonmetal ions.
  • molecular recognition complex refers to any molecule, molecular group, or molecular complex that is capable of recognizing (i.e., specifically interacting with) a molecule.
  • the ligand binding site of a receptor would be considered a molecular recognition complex.
  • the term “ambient condition” refers to the conditions of the surrounding environment (e.g., the temperature of the room or outdoor environment in which an experiment occurs).
  • room temperature refers, technically, to temperatures approximately between 20 and 25 degrees centigrade. However, as used generally, it refers to the any ambient temperature within a general area in which an experiment is taking place.
  • home testing and “point of care testing” refer to testing that occurs outside of a laboratory environment. Such testing can occur indoors or outdoors at, for example, a private residence, a place of business, public or private land, in a vehicle, under water, as well as at the patient's bedside.
  • lipid refers to a variety of compounds that are characterized by their solubility in organic solvents. Such compounds include, but are not limited to, fats, waxes, steroids, sterols, glycolipids, glycosphingolipids (including gangliosides), phospholipids, te ⁇ enes, fat-soluble vitamins, prostaglandins, carotenes, and chlorophylls.
  • lipid-based materials refers to any material that contains lipids.
  • virus refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell.
  • the individual particles consist of nucleic acid and a protein shell or coat; some virions also have a lipid containing membrane.
  • the term "virus” encompasses all types of viruses, including animal, plant, phage, and other viruses.
  • free floating aggregates refers to aggregates that are not immobilized.
  • encapsulate refers to the process of encompassing, encasing, or otherwise associating two or more materials such that the encapsulated material is immobilized within or onto the encapsulating material.
  • optical transparency refers to the property of matter whereby the matter is capable of transmitting light such that the light can be observed by visual light detectors (e.g., eyes and detection equipment).
  • biologically inert refers to a property of material whereby the material does not chemically react with biological material.
  • organic solvents refers to any organic molecules capable of dissolving another substance. Examples include, but are not limited to, chloroform, alcohols, phenols, and ethers.
  • nanostructures refers to microscopic structures, typically measured on a nanometer scale. Such structures include various three-dimensional assemblies, including, but not limited to, liposomes, films, multilayers, braided, lamellar, helical, tubular, and fiber-like shapes, and combinations thereof. Such structures can, in some embodiments, exist as solvated polymers in aggregate forms such as rods and coils.
  • films refers to any material deposited or used in a thin section or in a layer form.
  • vesicle refers to a small enclosed structures. Often the structures are membranes composed of lipids, proteins, glycolipids, steroids or other components associated with membranes. Vesicles can be naturally generated (e.g., the vesicles present in the cytoplasm of cells that transport molecules and partition specific cellular functions) or can be synthetic (e.g., liposomes).
  • liposome refers to artificially produced spherical lipid complexes that can be induced to segregate out of aqueous media.
  • biopolymeric liposomes refers to liposomes that are composed entirely, or in part, of biopolymeric material.
  • tubules refers to materials comprising small hollow cylindrical structures.
  • solvated polymer As used herein, the terms “solvated polymer,” “solvated rod,” and “solvated coil” refer to polymerized materials that are soluble in aqueous solution.
  • multilayer refers to structures comprised of two or more monolayers.
  • the individual monolayers may chemically interact with one another (e.g., through covalent bonding, ionic interactions, van der Waals” interactions, hydrogen bonding, hydrophobic or hydrophilic assembly, and stearic hindrance) to produce a film with novel properties (i.e., properties that are different from those of the monolayers alone).
  • self-assembling monomers and “lipid monomers” refer to molecules that spontaneously associate to form molecular assemblies. In one sense, this can refer to surfactant molecules that associate to form surfactant molecular assemblies.
  • self-assembling monomers includes single molecules (e.g., a single lipid molecule) and small molecular assemblies (e.g., polymerized lipids), whereby the individual small molecular assemblies can be further aggregated (e.g., assembled and polymerized) into larger molecular assemblies.
  • “Surfactant molecular assemblies” refers to an assembly of surface active agents that contain chemical groups with opposite polarity, form oriented monolayers at phase interfaces, form micelles (colloidal particles in aggregation colloids), and have detergent, foaming, wetting, emulsifying, and dispersing properties.
  • the term “homopolymers” refers to materials comprised of a single type of polymerized molecular species.
  • mixtureed polymers refers to materials comprised of two or more types of polymerize molecular species.
  • ligands refers to any ion, molecule, molecular group, or other substance that binds to another entity to form a larger complex. Examples of ligands include, but are not limited to, peptides, carbohydrates, nucleic acids, antibodies, or any molecules that bind to receptors.
  • the term “dopant” refers to molecules that are added to biopolymeric materials to change the material's properties.
  • Dopant materials include, but are not limited to, lipids, cholesterols, steroids, ergosterols, polyethylene glycols, proteins, peptides, or any other molecule (e.g., surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, zwitterionic detergents, decylglucoside, deoxycholate. diacetylene derivatives, phosphatidylserine, phosphatidylinositol.
  • phosphatidylethanolamine phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cerebroside, lysophosphatidylcholine, D-erythroshingosine, sphingomyelin, dodecyl phosphocholine, N- biotinyl phosphatidylethanolamine. and other synthetic or natural components of cell membranes) that can be associated with a membrane (e.g., liposomes and films).
  • organic matrix and “biological matrix” refer to collections of organic molecules that are assembled into a larger multi-molecular structure. Such structures can include, but are not limited to, films, monolayers, and bilayers.
  • organic monolayer refers to a thin film comprised of a single layer of carbon-based molecules. In one embodiment, such monolayers can be comprised of polar molecules whereby the hydrophobic ends all line up at one side of the monolayer.
  • monolayer assemblies refers to structures comprised of monolayers.
  • organic polymetric matrix refers to organic matrices whereby some or all of the molecular constituents of the matrix are polymerized.
  • head group and “head group functionality” refer to the molecular groups present an the ends of molecules (e.g., the carboxylic acid group at the end of fatty acids).
  • hydrophilic head-group refers to ends of molecules that are substantially attracted to water by chemical interactions including, but not limited to, hydrogen-bonding, van der Waals' forces, ionic interactions, or covalent bonds.
  • hydrophobic head-group refers to ends of molecules that self-associate with other hydrophobic entities, resulting in their exclusion from water.
  • carboxylic acid head groups refers to organic compounds containing one or more carboxyl (-COOH) groups located at, or near, the end of a molecule.
  • carboxylic acid includes carboxyl groups that are either free or exist as salts or esters.
  • detecting head group refers to the molecular group contained at the end of a molecule that is involved in detecting a moiety (e.g., an analyte).
  • linker or "spacer molecule” refers to material that links one entity to another.
  • a molecule or molecular group can be a linker that is covalent attached two or more other molecules (e.g., linking a ligand to a self-assembling monomer).
  • polymeric assembly surface refers to polymeric material that provides a surface for the assembly of further material (e.g., a biopolymeric surface of a film or liposome that provides a surface for attachment and assembly of ligands).
  • the term "formation support” refers to any device or structure that provides a physical support for the production of material.
  • the formation support provides a structure for layering and/or compressing films.
  • diacetylene monomers refers to single copies of hydrocarbons containing two alkyne linkages (i.e., carbon/carbon triple bonds).
  • standard trough and “standard Langmuir-Blodgett trough” refer to a device, usually made of teflon, that is used to produce Langmuir films.
  • the device contains a reservoir that holds an aqueous solution and moveable barriers to compress film material that are layered onto the aqueous solution (See e.g., Roberts, Langmuir-Blodgett Films, Plenum, New York, [1990]).
  • crystalline mo ⁇ hology refers to the configuration and structure of crystals that can include, but are not limited to, crystal shape, orientation, texture, and size.
  • domain boundary refers to the boundaries of an area in which polymerized film molecules are homogeneously oriented.
  • a domain boundary can be the physical structure of periodic, regularly arranged polydiacetylene material (e.g., striations, ridges, and grooves).
  • domain size refers to the typical length between domain boundaries.
  • conjugated backbone and “polymer backbone” refer to the ene-yne polymer backbone of polymerized diacetylenic films that, on a macroscopic scale, appears in the form of physical ridges or striations.
  • polymer backbone axis refers to an imaginary line that runs parallel to the conjugated backbone.
  • intrabackbone and “interbackbone” refer to the regions within a given polymer backbone and between polymer backbones, respectively. The backbones create a series of lines or “linear striations,” that extend for distances along the template surface.
  • bond refers to the linkage between atoms in molecules and between ions and molecules in crystals.
  • single bond refers to a bond with two electrons occupying the bonding orbital. Single bonds between atoms in molecular notations are represented by a single line drawn between two atoms (e.g., C 8 -C 9 ).
  • double bond refers to a bond that shares two electron pairs. Double bonds are stronger than single bonds and are more reactive.
  • triple bond refers to the sharing of three electron pairs.
  • ene-yne refers to alternating double and triple bonds.
  • amine bond refers to any bond formed between an amine group (i.e.. a chemical group derived from ammonia by replacement of one or more of its hydrogen atoms by hydrocarbon groups), a thiol group (i.e., sulfur analogs of alcohols), and an aldehyde group (i.e., the chemical group -CHO joined directly onto another carbon atom), respectively, and another atom or molecule.
  • amine group i.e. a chemical group derived from ammonia by replacement of one or more of its hydrogen atoms by hydrocarbon groups
  • a thiol group i.e., sulfur analogs of alcohols
  • aldehyde group i.e., the chemical group -CHO joined directly onto another carbon atom
  • abso ⁇ tion refers, in one sense, to the abso ⁇ tion of light. Light is absorbed if it is not reflected from or transmitted through a sample. Samples that appear colored have selectively absorbed all wavelengths of white light except for those corresponding to the visible colors that are seen.
  • the term “spectrum” refers to the distribution of light energies arranged in order of wavelength.
  • visible spectrum refers to light radiation that contains wavelengths from approximately 360 nm to approximately 800 nm.
  • ultraviolet irradiation refers to exposure to radiation with wavelengths less than that of visible light (i.e., less than approximately 360 nM) but greater than that of X-rays (i.e., greater than approximately 0.1 nM). Ultraviolet radiation possesses greater energy than visible light and is therefore, more effective at inducing photochemical reactions.
  • chromatic transition refers to the changes of molecules or material that result in an alteration of visible light abso ⁇ tion.
  • chromatic transition refers to the change in light abso ⁇ tion of a sample, whereby there is a detectable color change associated with the transition. This detection can be accomplished through various means including, but not limited to, visual observation and spectrophotometry.
  • thermochromic transition refers to a chromatic transition that is initiated by a change in temperature.
  • solid support refers to a solid object or surface upon which a sample is layered or attached.
  • Solid supports include, but are not limited to, glass, metals, gels, and filter paper, among others.
  • Hydrodrophobized solid support refers to a solid support that has been chemically treated or generated so that it attracts hydrophobic entities and repels water.
  • film-ambient interface refers to a film surface exposed to the ambient environment or atmosphere (i.e., not the surface that is in contact with a solid support).
  • formation solvent refers to any medium, although typically a volatile organic solvent, used to solubilize and distribute material to a desired location (e.g., to a surface for producing a film or to a drying receptacle to deposit liposome material for drying).
  • the term “micelle” refers to a particle of colloidal size that has a hydrophilic exterior and hydrophobic interior.
  • topochemical reaction refers to reactions that occur within a specific place (e.g., within a specific portion of a molecule or a reaction that only occurs when a certain molecular configuration is present).
  • the term “molding structure” refers to a solid support used as a template to design material into desired shapes and sizes.
  • array and “patterned array” refer to an arrangement of elements (i.e., entities) into a material or device. For example, combining several types of biopolymeric material with different analyte recognition groups into an analyte-detecting device, would constitute an array.
  • interferants refers to entities present in an analyte sample that are not the analyte to be detected and that, preferably, a detection device will not identify, or would differentiate from the analyte(s) of interest.
  • the term "badge” refers to any device that is portable and can be carried or worn by an individual working in an analyte detecting environment.
  • the term “device” refers to any apparatus (e.g., multi-well plates and badges) that contain biopolymeric material.
  • the biopolymeric material may be immobilized or entrapped in the device. More than one type of biopolymeric material can be inco ⁇ orated into a single device.
  • the term “halogenation” refers to the process of inco ⁇ orating or the degree of inco ⁇ oration of halogens (i.e., the elements fluorine, chlorine, bromine, iodine and astatine) into a molecule.
  • aromaticity refers to the presence of aromatic groups (i.e., six carbon rings and derivatives thereof) in a molecule.
  • water-immiscible solvents refers to solvents that do not dissolve in water in all proportions.
  • water-miscible solvents refers to solvents that dissolve in water in all proportions.
  • positive and negative refer to molecules or molecular groups that contain a net positive, negative, or neutral charge, respectively. Zwitterionic entities contain both positively and negatively charged atoms or groups whose charges cancel (i.e., whose net charge is 0).
  • biological organisms refers to any carbon-based life forms.
  • aqueous refers to a liquid mixture containing water, among other components.
  • solid-state refers to reactions involving one or more rigid or solid-like compounds.
  • the term “regularly packed” refers to the periodic arrangement of molecules within a compressed film.
  • filtration refers to the process of separating various constituents within a test sample from one another.
  • filtration refers to the separation of solids from liquids or gasses by the use of a membrane or medium.
  • the term encompasses the separation of materials based on their relative size.
  • inhibitor refers to a material, sample, or substance that retards or stops a chemical reaction.
  • reaction means inhibitor refers to inhibitors that are capable of retarding or stopping the action or activity of a given reaction means (e.g., an enzyme).
  • inhibitor screening refers to any method used to identify and/or characterize inhibitors.
  • inhibitor screening methods provide "high throughput screening,” the ability to screen a large number of samples suspected of containing inhibitors in a short period of time. It may also be desired that the inhibitor screening method provide quantifiable results to provide comparisons of inhibitor efficiency.
  • sample is used in its broadest sense. In one sense it can refer to a biopolymeric material. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • the present invention relates to methods and compositions for the direct detection of membrane conformational changes through the detection of color changes in biopolymeric materials.
  • the present invention allows for the direct colorimetric detection of membrane modifying reactions and analytes responsible for such modifications and for the screening of reaction inhibitors.
  • the materials undergo a detectable (e.g., visually detectable) color change.
  • the present invention provides for the direct colorimetric detection of a variety of membrane disrupting events including, but not limited to, lipid cleavage, polymerization, lipid flipping, transmembrane signalling, vesicle formation, lipidation, glycosylation, ion channeling, molecular rearrangement, and phosphorylation among others.
  • lipid cleavage lipid cleavage
  • polymerization lipid flipping
  • transmembrane signalling vesicle formation
  • lipidation glycosylation, ion channeling
  • molecular rearrangement phosphorylation among others.
  • results can be inte ⁇ reted by an untrained observer, and the methods can be conducted under ambient conditions, making them amenable to numerous uses including, but not limited to. home testing diagnostics, field work, detection of air-borne or water-borne materials, military applications, doctor's office or point of care testing, and many other applications.
  • the present invention provides detecting technology that does not require an energy source and is cost-efficient, stable, accurate, reliable, consistent, and robust. These enhanced qualities provide an ideal basis for use in screening new compound libraries (e.g., drug screens), identification and characterization of enzyme inhibitors, drug testing, water supply testing, and any application in which a rapid and accurate colorimetric screen is desired.
  • new compound libraries e.g., drug screens
  • identification and characterization of enzyme inhibitors e.g., drug testing, water supply testing, and any application in which a rapid and accurate colorimetric screen is desired.
  • the biopolymeric materials of the presently claimed invention offer a one-step approach to measuring enzyme activity through detection of a color change of diacetylene 'signaling' lipids that surround the natural enzyme substrate.
  • the strategy does not require additional chemical reagents or post-hydrolysis analytical methods.
  • enzyme inhibitors can be rapidly identified by simply monitoring the color changes of aqueous vesicle suspensions in a standard 96-well microtiter plate or equivalent.
  • Conjugated polymers (CPs) such as polydiacetylene (PDA), polythiophene, and polypyrrole display a remarkable array of color transitions arising from thermal changes (thermochromism) (Levesque and Leclerc, Chem. Mater.
  • the methods and compositions of the presently claimed invention provide a visually detectable colorimetric change in the polymerized materials and do not require the use of a transducing device.
  • the presently claimed invention provides novel biochromatic detection methods comprising chemical modification of PDA-vesicles by interfacial enzymes such as phospholipase A 2 (PLA 2 ). These methods offers a new pathway of inducing the biochromic effect.
  • the color change of the vesicle solution is driven by hydrolysis of a natural, unlabeled enzyme substrate embedded in the PDA matrix.
  • the presently claimed invention demonstrates that the biochromatic transition of the PDA vesicles is suppressed by the addition of a known phospholipase inhibitor, providing applications in high throughput drug discovery.
  • the present invention also provides an array of biopolymeric materials inco ⁇ orated into a single device, such that each individual section of biopolymeric material respond differently to different reactions or to a given reaction.
  • arrays can be designed so that the presence of a certain reaction will produce a color change in a known location in the device, or that will produce a color change specific to the given reaction (e.g., pu ⁇ le to orange for reaction X and blue to red for reaction Y). It is also contemplated that other arrays will be used with the present invention, including such easily understood patterns as a "+" sign to indicate that presence of a particular substance, compound, or reaction. It is not intended that the present invention be limited to any particular array design or configuration.
  • the present invention provides methods and compositions for the characterization of membrane rearrangements that overcome many of the disadvantages of currently available technologies (e.g., indirect detection, sample purification, cost, and use of radioactivity or other hazardous materials).
  • the presently claimed invention comprises methods and compositions related to biopolymeric materials that change color in response to membrane rearrangements (e.g., lipid cleavage).
  • biopolymeric materials comprise many forms including, but not limited to, films, vesicles, tubules, multilayered structures, and solvated rods and coils. These materials are comprised of polymerized self-assembling monomers.
  • the biopolymeric materials comprise more than one species of self-assembling monomer. Some of these self-assembling monomers may lack polymerizable groups.
  • the materials further comprise dopant material(s) that alter the properties of the sensor.
  • Dopants include, but are not limited to, polymerizable self-assembling monomers, non- polymerizable self-assembling monomers, lipids, sterols, membrane components, and any other molecule that optimizes the biopolymeric material (e.g., material stability, durability, colorimetric response, and immobilizability).
  • the biopolymeric material may further comprise ligands (e.g., proteins, antibodies, carbohydrates, and nucleic acids).
  • the ligands can provide attachment sites for recruiting molecules to the biopolymeric surface or can be used as binding sites for analytes. whereby the binding event causes a colorimetric change in the biopolymeric material.
  • the various embodiments of the presently claimed invention provide the ability to colorimetrically detect a broad range reactions and analytes. With certain biopolymeric materials, a color transition in response to a reaction can be viewed by simple visual observation or, if desired, by color sensing equipment.
  • the present invention further provides a variety of means of immobilizing the biopolymeric material to provide stability, durability, and ease of handling and use.
  • a variety of different polymeric materials are combined into a single device to produce an array.
  • the array can be designed to detect and differentiate differing types or quantities of reactions or analytes (i.e., the array can provide quantitative and/or qualitative data).
  • the methods and compositions of the presently claimed invention find use in a broad range of analyte detection circumstances and are particularly amenable to situations where simple, rapid, accurate, and cost-efficient detection is required.
  • biopolymeric materials described in these sections can be designed to detect the presence of analytes (e.g., pathogens, chemicals, and proteins) or can be designed to detect membrane rearrangements (e.g., lipid cleavage events). In some embodiments, it may be desired to have biopolymeric materials that accomplish both of these functions.
  • the optimization of the biopolymeric materials e.g., optimization of colorimetric response, color, and stability
  • the detection of analytes or membrane rearrangements is often generally applicable to both scenarios. Where there are differences, it is noted.
  • the biopolymeric material of the presently invention can take many physical forms including, but not limited to, liposomes, films, and multilayers, as well as braided, lamellar, helical, tubular, and fiber-like shapes, and combinations thereof.
  • the biopolymeric materials are solvated polymers in aggregate forms such as rods and coils.
  • the biopolymeric material used in the presently claimed invention comprise biopolymeric film.
  • biopolymeric films were prepared by layering the desired matrix-forming material (e.g., self-assembling organic monomers) onto a formation support.
  • the formation support was a standard Langmuir-Blodgett trough and the matrix-forming material was layered onto an aqueous surface created by filling the trough with an aqueous solution. The material was then compressed and polymerized to form a biopolymeric film.
  • the compression was conducted in a standard Langmuir-Blodgett trough using moveable barriers to compress the matrix-forming material. Compression was carried out until a tight- packed monolayer of the matrix- forming material was formed. Films provide a very sensitive colorimetric screen for analytes.
  • the matrix- forming material located within the formation support, was polymerized by ultra-violet irradiation. All methods of polymerization are contemplated by the present invention and include, but are not limited to, gamma irradiation, x-ray irradiation, chemical crosslinking, and electron beam exposure.
  • diacetylene monomers (DA) were used as the self-assembling monomer.
  • the diacetylene monomers (DA) were polymerized to polydiacetylene (p-PDA or PDA) using ultraviolet irradiation.
  • the ultraviolet radiation source is kept sufficiently far from the film to avoid causing heat damage to the film.
  • the crystalline mo ⁇ hology of the polymerized film can be readily observed between crossed polarizers in an optical microscope, although this step is not required by the present invention.
  • the visibly blue films were then transferred to hydrophobized solid supports, such that the carboxylic acid head groups were exposed at the film-ambient interface (Charych et al. Science 261 : 585 [1993]) to undergo further analysis, although the method of the present invention does not require this step.
  • Linear striations typical of PDA films can be observed in the polarizing optical microscope.
  • the material may also be characterized using atomic force microscopy or other characterization means (See e.g.,
  • films can be made by solvent casing (i.e., slow evaporation of the solvent).
  • lipid monomers can be made with silane or thiol anchoring groups, which allows dipping of solid supports into the solution to form a coated solid support. Diacetylene monomers are anchored by the silane and thiol groups and are then polymerized. This method eliminates the need for a trough.
  • the biopolymeric material used in the presently claimed invention comprises biopolymeric liposomes.
  • Liposomes were prepared using a probe sonication method (New, Liposomes: A Practical Approach, Oxford University Press, Oxford, pp 33-104 [1990]), although any method that generates liposomes is contemplated.
  • Self-assembling monomers either alone, or associated with a desired ligand, were dried to remove the formation solvents and resuspended in deionized water. The suspension was probe sonicated and polymerized. The resulting liposome solution contained biopolymeric liposomes. Liposomes differ from monolayers and films in both their physical characteristics and in the methods required to generate them.
  • Monolayers and films (or multilayers) made from amphiphilic compounds are planar membranes and form a two-dimensional architecture.
  • Monolayers and films in this context, are solid state materials that are supported by an underlying solid substrate as shown in Figure 1.
  • Film Y is a centrosymmetric multilayer film
  • films X and Z are noncentrosymmetric multilayers.
  • Ulman Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self- Assembly, Academic Press, Inc., Boston, [1991]
  • Gaines Gaines. Insoluble Monolayers at Liquid-Gas Interfaces, Interscience Publishers, New York, [1966]).
  • liposomes are three-dimensional vesicles that enclose an aqueous space as shown in Figure 2.
  • Figure 2 shows A) a cross-section two-dimensional view; and B) a three-dimensional view of half of a liposome.
  • Liposomes and monolayers do not enclose an aqueous space and do not entrap materials within a compartment.
  • the liposomes are typically more stable and robust than the films made of the same material.
  • Liposomes and films are prepared using different methods. Liposomes are prepared by dispersal of amphiphilic molecules in an aqueous media and remain in the liquid phase. In contrast, monolayers and films are prepared by immobilizing amphiphilic molecules at the air-water interface. A solid support is then passed through the interface to transfer the film to the solid support. Liposomes exist within homogenous aqueous suspensions and may be created in a variety of shapes such as spheres, ellipsoids, squares, rectangles, and tubules.
  • liposomes resemble the three-dimensional architecture of natural cell membranes. If liposomes are dried to their solid state, they may lose their shape and no longer exist in a liposomal state (i.e., are no longer "liposomes"). In contrast, films exist as planar heterogeneous coatings, immobilized onto a solid support. The surface of a monolayer or film can be in contact with air, other gases, or other liquids.
  • Films can be dried in air and maintain their planar monolayer or multilayer structure and thus remain as "films."
  • a much higher concentration of polymerized material can be achieved with liposome solutions compared to monolayer assemblies, due to their greater cross-sectional density.
  • Liposomes have the advantage, generally, of making the color change more visually striking and increasing the colorimetric response (See e.g., Figure 3 showing the colorimetric response of immobilized sialic-acid-containing liposomes (1) and films (2) to the presence of influenza virus).
  • liposomes of the present invention In designing methods to generate the liposomes of the present invention, several difficulties had to be overcome. While it was initially hoped that liposomes could be generated with the self-assembling monomer material (e.g., diacetylenes) used in various film embodiments (i.e., film embodiments of the present invention discussed above and in
  • Example 1 it was not known whether this would be possible, largely due to the differences in liposomal and film architecture. Liposomes are three-dimensional instead of two- dimensional. Therefore, it was not clear whether 1 ) the diacetylenic lipids would actually form liposomes at all; 2) whether they would polymerize if they were capable or forming liposomes; and/or 3) whether they would exhibit colorimetric properties even if they could be polymerized.
  • the double chain molecules typically used in liposome formation are derived from natural cell membranes and usually have a classical phospholipid structure inco ⁇ orating such molecular components as phosphodiglycerides and sphingolipids, unlike the diacetylenic lipids of the present invention.
  • attempts to form liposomes with diacetylenic lipids using standard methods such as vortexing or bath sonications were tried (i.e., methods that are similar to those commonly applied to phospholipids). These methods failed to form liposomes and resulted in the formation of an insoluble, non-dispersed, non-characterizable mixture. This mixture did not exhibit colorimetric properties.
  • T m main phase transition temperature
  • Figure 4 shows a heating curve depicting the large main phase transition for unpolymerized liposomes prepared from lysine- derivated PDA monomer. Therefore, it was necessary to employ higher energy methods such as ultrasonic probe sonication and heating, to raise the temperature above T m and to disperse the lipid. Under these conditions (e.g., as described in Example 1) liposomes were formed, as evidenced by light scattering and transmission electron microscopy with a size in accordance with a liposome (i.e.. approximately 100 nm).
  • polymerization requires that the lipids pack in a precise distance and orientation with respect to one another.
  • the polymerization of polydiacetylene is therefore a "solid state" or topochemical polymerization. This is why the molecules must be closely packed to allow cross-linking.
  • This precise packing can be controlled in monolayer and films at the air-water interface using moveable barriers of Langmuir apparatus that can compress the film to the desired packing as shown in Figure 5, in which a compressed film is being transferred to a vertical plate.
  • no such external compression is possible.
  • the lipids assemble and occupy an equilibrium distance and orientation with respect to one another. Therefore, prior to the development of the present invention, it was not clear that the distance and packing between the molecules in the liposome material would be sufficient to allow the polymerization reaction to take place.
  • the most difficult aspect was cross-linking the liposome diacetylenic monomeric lipids, to generate a polydiacetylene conjugated polymer (i.e., polymerized liposomes). It is the conjugated polymer backbone that provides the liposomes with the desired color, and potentially allows the detection of biological analytes through an observable color change produced by the binding of the analyte to the liposomes.
  • the liposomes were formed (i.e., using the methods described above) and cooled to room temperature, it was found that they did not polymerize at all upon exposure to ultraviolet light.
  • lipids should have crystallized and returned to their solid-like state when cooled to room temperature (i.e., once the lipids returned to this state, they should have undergone the topochemical polymerization as described above). However, they did not, as apparently the lipids were still fluid. Further analysis by transmission electron microscopy (TEM) proved that the liposomes were not crystallized. These room temperature liposomes aggregated into larger globules, characteristic of non-stabilized fluid phase liposomes as shown in the micrograph of Figure 6. Based upon these observations, it was hypothesized that there was a hysteresis effect in the heating/cooling curve of these materials.
  • TEM transmission electron microscopy
  • nanostructures include, but are not limited to, multilayers, braided, lamellar, helical, tubular, and fiber-like shapes, and combinations thereof.
  • Such structures can, in some embodiments, be solvated polymers in aggregate forms such as rods and coils. For example, it has been shown that the chain length of the monomers effects the type of aggregate that forms in solution (Okahata and Kunitake, J. Am. Chem. Soc. 101 : 5231 [1979]).
  • soluble polymers of polythiophenes can be generated.
  • sugar groups, peptides, or other ligands can be synthesized as thiophene derivatives and then polymerized as co-polymers.
  • NHS derivatives of thiophene can be polymerized and ligand groups can be attached after the polymer has formed (described below).
  • the thiophene polymers are rendered water soluble by the addition of acid groups. Thus they can be synthesized to freely dissolve in aqueous solution, creating a colorimetric solution.
  • the present invention contemplates a variety of self-assembling monomers that are suitable for formation of biopolymeric materials.
  • Such monomers include, but are not limited to, acetylenes, diacetylenes (e.g., 5,7-docosadiynoic acid, 5,7-pentacosadiynoic acid, and 10,12-pentacosadiynoic acid), alkenes, thiophenes, polythiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes, poly-silanes, anilines, pyrroles, polyacetylenes, poly (para- phylenevinylene).
  • acetylenes e.g., 5,7-docosadiynoic acid, 5,7-pentacosadiynoic acid, and 10,12-pentacosadi
  • the biopolymeric material of the present invention may comprise a single species of self-assembling monomer (e.g., may be made entirely of 5,7-pentacosadiynoic acid) or may comprise two or more species.
  • solvents containing the individual monomers are combined in the desired molar ratio.
  • This mixture is then prepared as described above (e.g., layering onto the aqueous surface of a Langmuir-Blodgett device for film preparation or evaporated and resuspended in aqueous solution for liposome preparation).
  • the self- assembling monomers may be chemically linked to another molecule (e.g., a ligand).
  • diacetylene monomers are used as the self-assembling monomers of the biopolymeric material of the present invention.
  • the present invention contemplates a variety of diacetylenes including, but not limited to 5,7-docosadiynoic acid
  • the presently claimed invention further contemplates the optimization of the biopolymeric material to maximize response to given reaction conditions.
  • the chemistry of the particular lipid used in the biopolymeric material plays a critical role in increasing or decreasing the sensitivity of the colorimetric transition. For example, a positional variation of the chromophoric polymer backbone can alter sensitivity to a given analyte.
  • such improved sensitivity allowed detection of small analytes (e.g., bacterial toxins such as cholera toxin from Vibrio cholerae and pertussis toxin, as well as antibodies). It is contemplated that further optimization will generate sensitive materials for the detection of many reactions, rearrangements, and analytes.
  • small analytes e.g., bacterial toxins such as cholera toxin from Vibrio cholerae and pertussis toxin, as well as antibodies.
  • the carbon chain length that positions the head group a specific distance from the polymer backbone in the final polymerized material is dependent on the position of the polymerizable group in an unassembled monomer.
  • diacetylene liposomes it has been shown that a diacetylene group positioned from between the 18-20 positions to the 3-5 position in the monomers produced progressively more sensitive liposomes when used for the detection of analytes. Liposomes produced from monomers with the diacetylene groups from the 10-12 position to the 4-6 position provides particularly efficient control of sensitivity.
  • Diacetylene groups positioned in about the 5-7 position are preferred for certain embodiments, such as cholera toxin detection.
  • the production protocol for the monomer determines at which position the diacetylene group will be placed in the final monomer product.
  • the total carbon chain length in the unassembled monomer also influences the level of sensitivity of the liposome product, although to a lesser extent than the position of the polymerizable group in the monomer carbon chain.
  • the shorter chain length typically provides for greater sensitivity for. as determined in analyte-detecting embodiments.
  • the monomers that are ideally useful in construction of the inventive colorimetric liposomes can range from between C, 2 to C 25 in length, although both longer and shorter chain lengths are contemplated by the presently claimed invention.
  • a preferred range of monomer carbon chain length in the present invention is C 20 to C 23 . The influence of monomer chain lengths and positioning of the polymerizable group on the chain has been demonstrated in several experiments.
  • C 23 chains provided a final colorimetric liposomes product that changed color at a lower analyte level than those produced from monomers with a C 25 chain.
  • the C 22 length chain provided a greater sensitivity than the C 24 length chain.
  • the chain length is designed so as to be suitable for the optimal detection conditions of interest.
  • the biopolymeric materials of the present invention may further comprise one or more dopant materials.
  • Dopants are included to alter and optimize desire properties of the biopolymeric materials. Such properties include, but are not limited to, colorimetric response, color, sensitivity, durability, robustness, amenability to immobilization, temperature sensitivity, and pH sensitivity.
  • Dopant materials include, but are not limited to, lipids, cholesterols, steroids, ergosterols, polyethylene glycols, proteins, peptides, or any other molecule (e.g., surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cerebroside, lysophosphatidylcholine, D-erythroshingosine, sphingomyelin, dodecyl phosphocholine, N- biotinyl phosphatidylethanolamine, and other synthetic or natural components of cell membranes
  • Example 4 demonstrate that the addition of sialic acid- derived diacetylene monomers to liposomes comprising ganglioside and PDA provided a dramatic increase in colorimetric sensitivity and quantifiability to the detection of low levels of analyte.
  • This improvement in colorimetric response using dopant is extremely beneficial when un-doped materials produce only weak signals.
  • the target lipids e.g., lipids that contain the ligand or that are the substrate of an enzymatic reaction
  • the polymer backbone e.g., ganglioside ligands
  • dopants are added to alter the color of the biopolymeric material.
  • the present invention provides liposomes that change from blue to red, but also blue to orange, pu ⁇ le to red, pu ⁇ le to orange, green to red, and green to orange.
  • glutamine derivatized PDA produced very dark blue (i.e., almost black) liposomes.
  • green liposomes were produced with cycles of annealing
  • the present invention provides a dopant cocktail that is a mix of glucose and sialic acid-derived polydiacetylene.
  • the glucose component of the dopant mixture appears to act primarily to prevent non-specific adhesion to the surface of the inventive liposome and may also enhance sensitivity.
  • the polydiacetylene bound sialic acid component appears to functionally destabilize the surface to provide a dramatic increase in sensitivity for analyte detection.
  • dopant lowers the activation barrier of the chromatic transition and/or provides a connection between the ligands (i.e., if ligands are present) and the conjugated backbone, enabling the reactions to induce the colorimetric transition.
  • dopants with bulky headgroups e.g., sialic acid-derived lipid monomers
  • dopants with bulky headgroups are subject to various solvent interactions at the matrix surface, destabilizing the structure of the blue film and thus allowing relatively small perturbations provided by the localized membrane rearrangements to complete the colorimetric transition.
  • the dopant comprises a diacetylene or a modified diacetylene (e.g., sialic acid derived diacetylene).
  • a diacetylene or a modified diacetylene e.g., sialic acid derived diacetylene
  • the derivatized lipid is used to modify the properties of the biopolymeric material and is not used as a molecular recognition site for an analyte detection (e.g., as in the case of sialic acid ligand used to detect influenza virus).
  • a diacetylene-based polymeric material containing only sialic acid derivatized monomer or lactose derivatized monomer did not respond to neurotoxins (e.g., botulinum neurotoxin), indicating that there was an insufficient interaction between the neurotoxins and the derivatized diacetylene lipid to induce the color change.
  • neurotoxins e.g., botulinum neurotoxin
  • a colorimetric response was detected in the presence of neurotoxin.
  • the sialic acid and lactose derived lipids are "dopants" and the ganglioside G M , is a ligand.
  • dopant materials will find use in optimizing the properties of the biopolymeric material used in various embodiments of the present invention.
  • Materials that are constituents of cell membrane structures in nature are generally useful as dopants in the present invention.
  • steroids e.g., cholesterols
  • Surfactant type compounds also may serve as dopants, whether or not they are polymerized to self-assembling monomers that make up the polymer back bone.
  • the detergent TWEEN 20 which does not contain a polymerizable group, has been shown to provide a very dramatic intensity to the blue color of the liposomes of certain embodiments of the present invention.
  • peptide-detergents i.e., small amphipathic molecules that have a hydrophobic region mimicking the membrane spanning regions of membrane proteins. These small peptides (typically 20-25 amino acids in length) can be inco ⁇ orated into the biopolymeric material to alter the stability or sensitivity of the colorimetric response of the material. Since peptide- detergents are bulkier in the hydrophobic region of the material, they are capable of producing a more pronounced effect on film stability or sensitivity than many other surfactant molecules. The most appropriate percentage of dopant inco ⁇ orated into the structure of the biopolymeric material is dependent on the particular system being developed, and the needs of the testing situation.
  • sensitivity may be compromised to some extent in the favor of long shelf life, or to accommodate rigorous field conditions.
  • the acceptable percentage of dopant is theoretically limited only to that which will not preclude sufficient inco ⁇ oration of the indicator polydiacetylene molecules to produce the necessary optical density and color change or to that which will disrupt the stability of the polymeric structures.
  • Molar percentages of dopant can vary from as low as 0.01% where increases of sensitivity have been observed in certain embodiments, to as high as 75%, after which the structural integrity of the biopolymeric material typically begins to deteriorate. However, there may be specific embodiments where the percentage of dopant is greater than 75% or lower than 0.01%. A preferred range for dopant is 2%-10%. In certain embodiments of the present invention, the optimal percentage of dopant is about 5% (See e.g., Example 4, section II).
  • cholera toxin For example, for the detection of cholera toxin, it was found that a film comprising 2% lactose-derivatized polydiacetylene (PDA), 5% ganglioside, and 93% PDA resulted in a strong blue to red color change when the film was incubated with the analyte.
  • PDA lactose-derivatized polydiacetylene
  • the inco ⁇ oration is very controlled, and requires several hours of processing. This relatively slow, gentle inco ⁇ oration method allows the inco ⁇ oration of comparatively large or complex dopant materials.
  • the sonication bath approach is only suitable when it is intended that a relatively low percentage of dopant is to be inco ⁇ orated.
  • the point probe method allows the inco ⁇ oration of a much higher percentage of dopant material over a shorter period of time, typically from one to ten minutes.
  • this method is typically limited to inco ⁇ oration of small to intermediate sized dopant materials.
  • the temperature chosen for inco ⁇ oration are selected based on the particular analytical system and liposome parameters desired. A practitioner will be able to select parameters such as pH, choice of dilutents, and other factors based on the particular system and desired characteristics of the biopolymeric material.
  • a series of derivatized polydiacetylene dopant molecules have been synthesized with a wide range of physical characteristics. These dopants are not the same as filler molecules typically observed in biological membranes (i.e., cholesterol, proteins, lipids, detergents). They differ in that they provide unique and specific functionality to a given sensor system. The design of several dopants that provide specific functionality to the non-synthetic embodiments are described below and in Example 4.
  • a simple system has been designed so that the PDA molecule can easily be derivatized.
  • the synthesis is shown in Figure 9.
  • 10,12-pentacosadiynoic acid is modified to amine-couple to any molecule with a free amino group. Since all amino acids have a free amino group (lysine has 2 free amino groups), the 20 amino acids were each placed on the head of PDA molecules.
  • Each one of the derivatized PDA molecules has special properties that allow special functionality to be inco ⁇ orated into the biopolymeric material. For example, glutamine-PDA doped materials were the most sensitive, most water soluble, and most consistent colorimetric sensors. The properties of some of the other amino acid-derivated PDA molecules are described in Example 4. The water solubility, ability to form films and liposomes, color, and colorimetric response for representative amino acid- derived diacetylenes is shown in Figure 10.
  • the biopolymeric materials of the present invention may further comprise one or more ligands.
  • Ligands can act as the recognition site in the biopolymeric materials for analytes or as anchors for recruiting molecules or localizing reactions to the biopolymeric surface.
  • a disruption of the polymer backbone of the biopolymeric material occurs, resulting in a detectable color transition.
  • biopolymeric material comprising a ligand (e.g., an antibody for a particular lipase) for the cleavage means can be placed in a device next to biopolymeric material that detects the cleavage means reaction itself (described below). In this manner, both the presence and the activity of the cleavage means are detected in a single device.
  • a ligand e.g., an antibody for a particular lipase
  • Ligands can be linked by a linking arm to the self-assembling monomers, directly linked to the monomers, inco ⁇ orated into the biopolymeric matrix prior to or during the polymerization process, or attached to the matrix following polymerization (e.g., by linking ligands to matrix constituents that contain head groups that bind to the ligands or through other means).
  • Figure 11 provides a schematic depiction of one embodiment of the present invention.
  • Compound 1 shows a receptor-binding ligand (i.e., sialic acid) attached to one terminal end of a spacer molecule.
  • the second terminal end of the spacer molecule is attached to one of several monomers (e.g., 10,12-pentacosadiynoic acid) that have been polymerized so as to form a chromatic detection element.
  • Compound 2 shows the 10,12- pentacosadiynoic acid without an attached ligand.
  • the ligand group of the present invention can be comprised of a wide variety of materials.
  • the main criterion is that the ligand have an affinity for the analyte of choice.
  • ligands include, but are not limited to, peptides, carbohydrates, nucleic acids, biotin, drugs, chromophores, antigens, chelating compounds, short peptides, pepstatin, Diels- Alder reagents, molecular recognition complexes, ionic groups, polymerizable groups, dinitrophenols, linker groups, electron donor or acceptor groups, hydrophobic groups, hydrophilic groups, antibodies, or any organic molecules that bind to receptors.
  • the biopolymeric material can be composed of combinations of ligand-linked and unlinked monomers to optimize the desired colorimetric response (e.g., 5% ligand-linked dicosadynoic acid [DCDA] and 95% DCDA). Additionally, multiple ligands can be inco ⁇ orated into a single biopolymeric matrix. As is clear from the broad range of ligands that can be used with the present invention, an extremely diverse group of analytes can be detected.
  • the self-assembling monomers are not associated with ligands, but are directly assembled, polymerized, and used as colorimetric sensors. Such biopolymeric materials can find use in the detection of certain classes of analytes including, but not limited to, volatile organic compounds (VOCs).
  • VOCs volatile organic compounds
  • ligands are inco ⁇ orated to detect a variety of pathogenic organisms including, but not limited to, sialic acid to detect HIV (Wies et al, Nature 333: 426 [1988]), influenza (White et al. Cell 56: 725 [1989]), Chlamydia (Infect. Imm. 57: 2378 [1989]), Neisseria meningitidis.
  • polio virus receptor to detect polio virus
  • fibroblast growth factor receptor to detect he ⁇ es virus
  • oligomannose to detect Escherichia coli
  • ganglioside G M to detect Neisseria meningitidis
  • antibodies to detect a broad variety of pathogens e.g., Neisseria gonorrhoeae. V. vulnificus, V. parahaemolyticus, V. cholerae, and V. alginolyticus).
  • lipids with a diverse range of compounds (e.g., carbohydrates, proteins, nucleic acids, and other chemical groups) are well known in the art.
  • carboxylic acid on the terminal end of lipids can be easily modified to form esters, phosphate esters, amino groups, ammoniums, hydrazines, polyethylene oxides, amides, and many other compounds.
  • These chemical groups provide linking groups for carbohydrates, proteins, nucleic acids, and other chemical groups (e.g., carboxylic acids can be directly linked to proteins by making the activated ester, followed by reaction to free amine groups on a protein to form an amide linkage).
  • carboxylic acids can be directly linked to proteins by making the activated ester, followed by reaction to free amine groups on a protein to form an amide linkage.
  • Examples of antibodies attached to Langmuir films are known in the art (See e.g., Tronin et al, Langmuir 11: 385 [1995]; and Vikholm et al, Langmuir 12: 3276 [1996]).
  • the methods of the present invention provide a system to easily attach protein molecules, including antibodies, to the surface of polydiacetylene thin films and liposomes, thereby providing biopolymeric materials with "protein" ligands.
  • ligands include, but are not limited to, peptides, proteins, lipoproteins, glycoproteins, enzymes, receptors, channels, and antibodies.
  • analyte e.g., enzyme substrate, receptor ligand, antigen, and other protein
  • a disruption of the polymer backbone of the biopolymeric material may occur, resulting in a detectable color change.
  • the present invention contemplates protein ligands that are inco ⁇ orated into the biopolymeric material and those chemically associated with the surface of the biopolymeric material (e.g., chemically linked to the surface head group of a monomer in the biopolymeric monomer).
  • the colorimetric change resulting from disruption of the biopolymeric material can be detected using many methods. In preferred embodiments of the presently claimed invention, a color shift was observed simply by visual observation. Thus, the present invention may be easily used by an untrained observer such as an at-home user.
  • spectral test equipment well known in the art is employed to detect changes in spectral qualities beyond the limits of simple visual observation, including optical density to a particular illuminating light wavelength.
  • a spectrometer the spectrum of the material was measured before and after analyte introduction, and the colorimetric response (%CR) was measured.
  • the spectrum was then taken following analyte exposure and a similar calculation was made to determine the B nnal .
  • the presently claimed invention can be, if desired, attached to a transducer device.
  • optical fibers See e.g., Beswick and Pitt, J. Colloid Interface Sci. 124: 146 [1988]; and Zhao and Reichert. Langmuir 8: 2785 [1992]
  • quartz oscillators See e.g., Furuki and Pu, Thin Solid Films 210: 471 [1992]; and Kepley et al, Anal. Chem. 64: 3191 [1992]
  • electrode surfaces See e.g., Miyasaka et al., Chem. Lett., p.
  • the present invention provides a double-check (i.e., confirmation method) by observation of color change in the material.
  • the biopolymeric materials of the present invention can be coated on thin PzT materials that oscillate at a resonance frequency, producing a microelectromechanical system (MEMS system).
  • MEMS system microelectromechanical system
  • alterations in the biopolymeric material can be detected as a change in resonant frequency with colorimetric change providing a confirmation of event.
  • Sensitivity can also be enhanced by coupling the lipid-polymer to a photoelectric device, colorimeter, or fiber optic tip that can read at two or more specific wavelengths.
  • the device can be linked to an alternative signalling device such as a sounding alarm or vibration to provide simple inte ⁇ retation of the signal.
  • analytes e.g., lipid cleavage activity of lipases and membrane modification activity of transferases
  • the biopolymeric materials of the presently claimed invention can be used to detect a large variety of analytes including, but not limited to, small molecules, microorganisms, membrane receptors, membrane fragments, volatile organic compounds (VOCs), enzymes, drugs, antibodies, and other relevant materials by the observation of color changes that occur upon analyte binding.
  • VOCs volatile organic compounds
  • the presently claimed invention works under very mild testing conditions, providing the ability to detect small biomolecules in a near natural state and avoiding the risks associated with modification or degradation of the analyte.
  • the presently claimed invention provides methods for detecting conformational alterations in the biopolymeric material by observation of colorimetric changes.
  • conformational changes can be caused by the binding of an analyte to a ligand (described above) and through the chemical modification of the biopolymeric material by chemical reactions (e.g., enzymatic catalysis).
  • the presently claimed invention provides a simple protocol using biopolymeric material and offers a practical approach to detecting interfacial catalysis, identifying inhibitors, and screening enzymes and other catalytic entities (e.g., catalytic antibodies) to characterize their catalytic capabilities.
  • These methods use natural, unlabeled substrates, and catalysis or inhibition is signaled by the presence or lack of a color transition of the surrounding lipid-polymer assembly.
  • the one-step nature of the technique allows for convenient adaptation to high throughput compound screening. This method is generally applicable to factors that affect enzyme recognition and activity, and influence membrane reorganization.
  • Polymerized mixed vesicles are highly stable against chemical and physical degradation and offer a convenient, economical alternative to enzymatic assays that employ radiolabled substrates.
  • the vesicle stock solutions described by the present invention have been stored for over six months without affecting the results of the assay.
  • PLA 2 activity has previously been studied in a variety of model membrane systems such as polymerized vesicles (Dua et al, J. Biol. Chem. 270, 263 [1995]), micelles (Reynolds et al. supra), and monolayers (Grainger et al, supra; and Mirsky et al. Thin Solid Films 284, 939 [1996]) using labeling techniques (e.g., radioactivity and fluorescence).
  • labeling techniques e.g., radioactivity and fluorescence.
  • the presently claimed invention provides biopolymeric materials inco ⁇ orating PLA 2 substrate lipids for the colorimetric detection of PLA 2 enzyme activity.
  • Biopolymeric materials were prepared with a combination of polymerizable matrix lipid (e.g., 10,12-tricosadiynoic acid) and various mole fractions (0-40%) of PLA 2 substrate lipid (e.g., dimyristoylphosphatidylcholine [DMPC]) as described in Examples 1 and 10.
  • PLA 2 substrate lipid e.g., dimyristoylphosphatidylcholine [DMPC]
  • the biopolymeric materials containing the PLA 2 substrate lipid were liposomes as shown in Figure 12.
  • This figure shows DMPC substrate in a diacetylenic lipid matrix before (top) and after (bottom) polymerization.
  • the vesicles appeared deep blue to the naked eye and absorbed maximally at around 620 nm, as shown in Figure 13 (solid line).
  • the suspension Upon addition of PLA 2 to the DMPC PDA vesicles, the suspension rapidly turned red (i.e., within minutes) and exhibited a maximum ab
  • the color change was modulated by altering the mole percentage of the natural lipid DMPC in the PDA vesicle as shown in Figure 14. A relative color change of 10% or more is clearly observed with the naked eye. Within minutes, liposomes containing greater than 20% DMPC exhibited strong colorimetric responses. Liposomes with low molar ratios of DMPC
  • PLA 2 activity was independently measured using a labeled lipid analog inco ⁇ orated into the PDA matrix, allowing simultaneous measurement of product formation and colorimetric response of the vesicles.
  • the analog used was thioester l,2-bis-(S-decanoyl)-l,2-dithio-sn-glycero-3- phosphocholine (DTPC).
  • Figure 16 features 31 P NMR spectra of the DMPC/PDA vesicles prior to the addition of PLA 2 (Figure 16A), and following the enzymatic reaction ( Figure 16B).
  • the relatively broad, anisotropic 3I P resonance from the intact vesicles, Figure 16A corresponds to the choline head-group of DMPC embedded in the PDA vesicles.
  • the observation of 3I P anisotropy in Figure 16A indicates that DMPC molecules are immobilized within the vesicle matrix.
  • the 31 P signal was shifted downfield as shown in Figure 16B.
  • FIG. 16B The position of the 31 P resonance in Figure 16B coincides with the shift observed for the water-solubilized lyso- myristoylphosphatidylcholine, the hydrolysis product of DMPC. Furthermore, Figure 16B shows that the 31 P resonance observed in the suspension of the enzyme-treated vesicles becomes significantly narrower than the 31 P signal from the initial DMPC/PDA vesicle, Figure 16A, indicating a higher mobility of the phosphate group following PLA 2 catalysis (Smith and Ekiel, Phosphorous-31 NMR, Principles and Applications, Academic Press, Orlando, pp 447 [1984]). This result suggests dissolution of the lysolipid reaction products following the enzymatic reaction. 'H NMR data indicating the appearance of a distinct lysolipid phase following the reaction with PLA further supported this description.
  • PDA vesicles demonstrating that the methodology described by the present invention is generally applicable. These phospholipases cleave the polar head group region of glycerophospholipids, whereas phospholipase A 2 cleaves the acyl ester bond exclusively at the 2-acyl position.
  • the assay test for phospholipase D and C were run under similar conditions as the
  • PLA 2 assays Both PLD and PLC activity were successfully detected by the liposomes assay.
  • the PLD assay yielded a final colorimetric response of approximately 55%. However, the shape of the response curve was more gradual than that of PLA 2 .
  • the PLC assay yielded a final colorimetric response of 60% and the response curve was similar to that of PLA 2 . NMR experiments further verified the occurrence of interfacial catalysis by PLC and PLD.
  • Bungarotoxin (BUTX) ⁇ -bungarotoxin, a snake toxin from Bungarus multicinctus, is known to destroy synaptic vesicles and inhibit acetylcholine release. It is classified as a PLA 2 toxin and is composed of two subunits: a 12-kDa subunit that exhibits PLA 2 activity and a 7.5-kDa subunit that shares sequence homology with protease inhibitors.
  • This bungarotoxin assay provides an example of a large molecular assembly possessing enzymatic properties that is capable of producing a colorimetric change in the biopolymeric materials.
  • additional bungarotoxin-detecting features for example, antibodies raised against bungarotoxin (i.e., ligands) can be incorporated onto the biopolymeric materials in addition to DMPC.
  • the present invention will find use in detecting, measuring, and characterizing the enzyme activities of many other systems including, but not limited to, lipolytic enzymes, acyltransferases, protein kinases. glycosidases. isomerases, ligases, polymerases, and proteinases, among others.
  • lipolytic enzymes can be free in solution, or be part of larger molecular aggregates, cells, and pathogens.
  • glycosidases can be detected to measure their activity or as indicators of the presence of a pathogen.
  • Sialidases such as neuraminidase are found on influenza virus, and other sialidases are associated with Salmonella.
  • the presence of the pathogens can be detected.
  • other detection elements e.g., sialic acid ligands for detection of influenza virus
  • Substrates can also be provided to produce detection systems for proteinases.
  • Candida albicans can be detected though its protease activity on pepstatin substrates.
  • anthrax spores from Bacillus anthracis can be detected by identifying laccase activity though its reaction with a substrate.
  • Laccases are multi-copper-containing enzymes that catalyze oxidative conversion of a variety of substrates, including phenols, poly- phenols, and aromatic amines. Specific substrates include vanillic acid, syringic acid, and 2- 2'-azino-bis(3-ethyl-benzthioazoline-6-sulfonic acid).
  • nucleic acids onto the biopolymeric material to test the activity of nucleotide polymerases (e.g., DNA polymerase).
  • nucleotide polymerases e.g., DNA polymerase.
  • these assay systems will find use in techniques for identifying and characterizing polymerase inhibitors. From these examples, it is clear that the biopolymeric materials of the present invention find use in the colorimetric detection of a broad array of membrane conformational changes and reactions.
  • the presently claimed invention provides methods for detecting the activity of enzymes and other molecules that alter the conformation of biopolymeric membranes. These methods can be expanded to provide an accurate, and fast screening technique for identifying and characterizing inhibitors of the activity responsible for the colorimetric change (e.g., identifying and characterizing protease inhibitors by subjecting candidate inhibitors to biopolymeric materials comprising the protein substrates for the enzymes).
  • the color change of the DMPC/PDA vesicles can be suppressed by using inhibitors to PLA 2 .
  • the vesicles remained in their blue phase upon addition of PLA 2 .
  • the vesicles also do not change color in the presence of other enzymes such as lysozyme and glucose oxidase, both of which produce a colorimetric response below 5% after more than an hour of incubation with the 40% DMPC/PDA vesicles.
  • enzymes such as lysozyme and glucose oxidase, both of which produce a colorimetric response below 5% after more than an hour of incubation with the 40% DMPC/PDA vesicles.
  • the specificity of the colorimetric response provides the necessary selectivity for high throughput screening of enzyme inhibitors.
  • biopolymeric material comprising a substrate for the enzyme being tested, are placed into a multi-chambered device (e.g., a 96-well plate). Each well is incubated with a sample suspected of containing an enzyme inhibitor. The enzyme is then added and the observation of a color change is detected. Successful inhibitors will partially or completely prevent the enzyme from producing a color change in the biopolymeric material. Appropriate control samples (e.g., a sample with no inhibitor and a sample with known inhibitor) are run with the assay to provide confidence in the results.
  • a multi-chambered device e.g., a 96-well plate.
  • biopolymeric materials of the presently claimed invention further provide methods for screening the efficacy and activity of "designed" proteins, peptides, and catalytic antibodies.
  • engineering enzymes There is much current activity in engineering enzymes to be stable under specific conditions of solvent and heat, among other conditions.
  • a simple, accurate screen of these engineered proteins can be conducted under a variety of test conditions.
  • inventive methods can be used to screen and characterize the reactions of catalytic antibodies.
  • the biopolymeric material of the present invention can be immobilized on a variety of solid supports, including, but not limited to polystyrene, polyethylene, teflon, silica gel beads, hydrophobized silica, mica, filter paper (e.g., nylon, cellulose, and nitrocellulose), glass beads and slides, gold and all separation media such as silica gel, sephadex, and other chromatographic media.
  • the biopolymeric materials are immobilized in silica glass using the sol-gel process.
  • Immobilization of the colorimetric biopolymeric materials of the present invention may be desired to improve their stability, robustness, shelf-life, colorimetric response, color, ease of use, assembly into devices (e.g., arrays), and other desired properties.
  • placement of colorimetric materials onto a variety of substrates surfaces can be undertaken to create a test method similar to the well-known and easy to use litmus paper test.
  • the reflective properties of nylon filter paper greatly enhance the colorimetric properties of the immobilized polydiacetylene liposomes. Filter paper also increases the stability of the liposomes due to the mesh size.
  • the liposome embodiment of the present invention has been loaded into the ink cartridge of a ink jet printer and used to print biopolymeric liposome material onto paper as though it were ink.
  • the liposome material present on the paper maintained its colorimetric properties.
  • This embodiment demonstrates the ease with which patterned arrays can be generated into any desired shape and size. By using multiple cartridges (e.g., using a color printer), patterned arrays can be generated with different biopolymeric materials.
  • sol-gel process has been used for entrapping organic molecules such as dyes and biomolecules in silica gels (See e.g., Avnir, Accounts Chem. Res. 28: 328 [1995]; Yamanaka et al, Am. Chem. Soc. 117: 9095 [1995]; Miller et al, Non-Cryst. Solids 202: 279 [1996]; and Dave et al, Anal. Chem. 66: 1120A [1994]), prior to the development of the present invention, immobilization of self-organized molecular aggregates (e.g., biopolymeric material, self-assembling monomer aggregates, and liposomes) was not realized in sol-gel materials.
  • self-organized molecular aggregates e.g., biopolymeric material, self-assembling monomer aggregates, and liposomes
  • Embodiments of the presently claimed invention provide for the successful immobilization of spherical, bilayer lipid aggregates, and liposomes using an aqueous sol-gel procedure.
  • These molecular structures, and particularly liposomes, composed of biological or biomimetic (i.e., mimics nature) lipids are fairly robust under aqueous conditions and ambient temperatures, but can easily degrade in the presence of organic solvents and high temperatures.
  • the sol-gel process provides a facile method of immobilizing molecular aggregates with no detectable structure modification, creating robust structures that are easily fabricated into any desired size or shape.
  • the silica sol-gel material was prepared by sonicating tetramethylorthosilicate, water, and hydrochloric acid under chilled conditions until a single phased solution was obtained.
  • metal oxides other than tetramethylorthosilicate, are contemplated by the present invention, so long as they facilitate the entrapment and form substantially transparent glass material.
  • metal oxides include, but are not limited to. silicates, titanates, aluminates, ormosils, and others. Buffer was then added to the acidic solution under cooling conditions.
  • the biopolymeric materials, generated as described above, were mixed into the buffered sol solution. This composite was poured into a desired molding structure and allowed to gel at ambient temperatures.
  • DCDA liposomes were inco ⁇ orated into sol-gel glass, although inco ⁇ oration of any biopolymeric structure is contemplated by the present invention.
  • sol-gel matrix that is compatible with fragile biopolymeric structures (i.e., liposomes) and maintains those physical properties that were observed in bulk solution. Additionally, it is contemplated that sol gel prepared materials of various thicknesses will possess unique sensitivities to analytes. Thicker films have a higher surface to volume ratio and therefore may require a higher concentration of analyte to trigger the chromatic transition.
  • the gelling conditions of the sol-gel preparation can be optimized by varying gelling temperatures, gel materials, and drying conditions to generate material with desired pore sizes. Varying the crosslink density of the material also provides control over pore size. Pore sizes from nanometers to hundreds of nanometers or greater are contemplated by the present invention. Some gels allow size-selective screening of undesired material while maintaining analyte access to the ligand. Also, the sol-gel technique allows structures to be formed that can be molded into any desirable shape, including, but not limited to, cartridges, coatings, monoliths, powders, and fibers.
  • the biopolymeric material can be attached to membranes of poly(ether urethanes) or polyacrylonitrile. These membranes are porous, hydrophilic and can be used for affinity separations or immunodiagnosis.
  • the liposomes of the present invention can be coupled to these membranes by first attaching an activating group such as imidizolyl-carbonyl, succinimido, FMP or isocyanate to the membrane which adds rapidly to nucleophiles (e.g., -NH 2 , -SH, or -OH groups) present in the liposomes.
  • nucleophiles e.g., -NH 2 , -SH, or -OH groups
  • materials which have an -SH functionality can also be immobilized directly to gold surfaces, particles, or electrodes via the thiol-gold bond.
  • a solution of the liposomes containing the -SH group are incubated with the clean gold surface in water for 12-24 hours with stirring at room temperature.
  • materials can be immobilized to silicon chips or silica gel (e.g., silicon dioxide) using the procedure described in Example 8.
  • materials containing - NH 2 functionalities can also be immobilized onto surfaces with standard glutaraldehyde coupling reactions that are often used with the immobilization of proteins.
  • liposomes can be attached through their carboxy groups to surfaces comprising polyethyleneimine, a branched polymer with free amine groups.
  • Certain embodiments of the presently claimed invention contemplate the generation of a large palette of polymerizable lipids with different headgroup chemistries, ligands, dopants, monomers or other properties within a single device to increase selectivity, sensitivity, quantitation, ease of use, and portability, among other desired characteristics and qualities.
  • array format By using the array format, several advantages can be realized that overcome the shortcomings of a single sensor approach. These include the ability to use partially selective sensors and to measure multicomponent samples. This offers the possibility of sensing a specific sample in the presence of an interfering background, or to monitor two or more samples of interest at the same time. The sensitivities of a given lipid to a given sample can be determined in order to generate identifiable finge ⁇ rints characteristic of each sample.
  • the response fingerprint orange/pink/pu ⁇ le/blue-pu ⁇ le would indicate the presence of sample X.
  • Arrays can be generated that measure both the presence and activity of samples. For example, when characterizing a certain enzyme, one portion of the array can provide analyte- detecting capabilities for the enzyme (e.g., by inco ⁇ orating a ligand that interacts with the enzyme), while another provides and enzyme activity assay (e.g., by including a substrate for the enzyme within the biopolymeric material).
  • Such arrays can be expanded for use in inhibitor screening techniques where each portion of the array provides quantitative or qualitative data, or provides a control experiment.
  • N normal
  • M molar
  • mM millimolar
  • ⁇ M micromolar
  • mol molecular weight
  • mmol millimoles
  • ⁇ mol micromol
  • nmol nanomoles
  • pmol picomoles
  • g grams
  • mg milligrams
  • micrograms (micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); ⁇ l (microliters); cm (centimeters); mm (millimeters); ⁇ m (micrometers); nm (nanometers); ⁇ Ci (microcurie); mN (millinewton); A (angstrom); kDa (kilodalton); ppm (parts per million); N (newton); °C (degrees Centigrade); wt% (percent by weight); aq.
  • the self-assembling monomers to be inco ⁇ orated into the liposomes were dissolved in solvent (e.g., chloroform for diacetylenes and methanol for ganglioside G M1 ).
  • solvent e.g., chloroform for diacetylenes and methanol for ganglioside G M1 .
  • solvent solutions were mixed in appropriate volumes in a brown vial (i.e., to prevent light interference during the upcoming drying steps) to achieve the desired lipid mixture (e.g., 5% by mole of G M1 , 95% diacetylenes) and a total lipid content of approximately 2 ⁇ mol.
  • the solvent was then evaporated by rotary evaporation or with a stream of nitrogen gas.
  • the dried lipids were resuspended in sufficient de-ionized water to produce a 1-15 mM solution of lipid.
  • the solution was then sonicated for 15-60 minutes with a probe sonicator (Fisher sonic dismembrator model 300, 50% output, microtip) as described by New (New, supra).
  • the solution was heated during the sonication (in most cases the sonicating process alone provides sufficient heat) to a temperature above the phase transition of the lipids used (typically 30-90 °C).
  • the resulting mixture was filtered through a 0.8 micromole nylon filter (Gelman) or through a 5 mm Millipore Millex-SV filter and cooled to 4°C for storage or was polymerized.
  • oxygen in the solution was removed by bubbling nitrogen through the sample for 5-10 minutes.
  • Polymerization of the stirred liposome solution was conducted in a 1 cm quartz cuvette with a small 254 nm UV-lamp (pen-ray, energy: 1600 microwatt cm 2 ) at a distance of 3 cm.
  • the chamber was purged with nitrogen during the polymerization to replace all oxygen and to cool the sample.
  • Polymerization times varied between 5 and 30 minutes depending on the desired properties (e.g., color, polymerization degree) of the liposomes.
  • the solution was placed in a UV-chamber, without purging, and exposed to 0.3-20 J/cnr of ultraviolet radiation, preferably 1.6 J/cm 2 , for 5-30 minutes.
  • polymerization was conducted in a multi-chambered plate (e.g., ELISA plate). Approximately 200 ⁇ l of sonicated liposome solution was placed in each well of the plate. The plate was placed under a UV lamp with the distance between the plate and the lamp kept at 3 cm. Irradiation times typically lasted for a minute. Prolonged irradiation resulted in formation of pink/purple liposomes, indicating that a color change was initiated by
  • Polydiacetylene films were formed in a standard Langmuir-Blodgett trough (See e.g., Roberts, Langmuir Blodgett Films, Plenum, New York [1990]).
  • the trough was filled with water to create a surface for the film.
  • Distilled water was purified with a millipore water purifier with the resistivity of 18.2 M-Ohm.
  • Diacetylene monomers e.g., 5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid [Farchan Laboratories], 5,7-pentacosadiynoic acid, combinations thereof, or other self assembling monomers
  • a solvent spreading agent e.g., spectral grade chloroform [Fisher]
  • Monomers prepared in the concentration range of 1.0 to 2.5 mM were kept at a temperature of 4°C in the dark, and were allowed to equilibrate at room temperature before being used in experiments.
  • the film was physically compressed using moveable barriers to form a tightly-packed monolayer of the self-assembling monomers.
  • the monolayer was compressed to its tightest packed form (i.e., until a film surface pressure of 20-40 mN/m was achieved).
  • the film was polymerized.
  • Certain embodiments (e.g., embodiments with dopants) of the present invention may require surface pressure compression greater or less than 20-40 mN/m.
  • Ultraviolet irradiation was used to polymerize the monomers, although other means of polymerization are available (e.g., gamma irradiation, x-ray irradiation, and electron beam exposure). Pressure was maintained on the film with the moveable barriers throughout the irradiation process at surface pressure of 20-40 mN/m. An ultraviolet lamp was placed 20 cm or farther from the film and trough. It was found that if the lamp is placed closer to the film, damage to the diacetylene film may occur due to the effects of heating the film. The film was exposed to ultraviolet light with a wavelength of approximately 254 nm for approximately one minute. The polymerization was confirmed by observing the blue color acquired upon polymerized diacetylene formation and detecting the linear striations typical of polymerized diacetylene films with a polarizing optical microscope.
  • Self-assembling monomers to be inco ⁇ orated into the tubules were dissolved in solvent, mixed together, evaporated, and resuspended in water as described above for liposomes. 1-10% by volume of ethanol was added to the solution, although other organic solvents are contemplated by the present invention. The solution was then sonicated (with heating if necessary), filtered, cooled, and polymerized as described above for liposomes.
  • Diacetylene films were prepared in a Langmuir Blodgett trough as described above using a combination of PDA monomers and sialic acid-derived PDA monomers.
  • the floating polymerized assembly was lifted by the horizontal touch method onto a glass slide previously coated with a self-assembled monolayer of octadecyltrichlorosilane (OTS) as described (Maoz and Sagiv, J. Colloid Interface Sci. 100: 465 [1984]).
  • OTS octadecyltrichlorosilane
  • the slide was then examined by optical microscopy with the use of crossed polarizers as described (Day and Lando, Macromolecules 13: 1478 [1980]).
  • the films were further characterized by angle-resolved x-ray photoelectron spectroscopy (XPS) and ellipsometry.
  • XPS x-ray photoelectron spectroscopy
  • the XPS results indicated that the amide nitrogen atoms and the carbonyl carbon atoms of the head groups were localized at the surface relative to the methylene carbons of the lipid chains, demonstrating that the sialoside head group was presented at the surface of the film.
  • Ellipsometric analysis of the polydiacetylene monolayer coated on HF-treated silicon indicated a film thickness of approximately 40 A, in agreement with the expected value based on molecular modeling.
  • the present invention provides a variety of different biopolymeric material forms (e.g., liposomes, films, tubules, etc.), with and without dopant materials, with a variety of ligands, and immobilized in a variety of forms.
  • biopolymeric material forms e.g., liposomes, films, tubules, etc.
  • dopant materials e.g., dopant materials, with a variety of ligands, and immobilized in a variety of forms.
  • the biopolymeric material of the presently claimed invention can comprise a sample of pure monomers (e.g., pure diacetylene) or can comprise mixed monomers (e.g., PDA with
  • Ganglioside G M or dopant. Optimization of the percent composition of mixed monomers can be undertaken to provide biopolymeric material with desired properties. An example of such optimization is provided below for the detection of an analyte (i. e. , cholera toxin) with a ganglioside ligand.
  • an analyte i. e. , cholera toxin
  • G M concentration combinations of ligand
  • PDA were tested. If too much ligand molecule was added (i.e., low concentration of polymerized lipid), the films were unstable and had high background. If the films had too much polymerized lipid molecule, they were too stable and the color change would not occur well.
  • search of the G M1 /PDA biosensor composition capable of displaying maximal response a series of PDA monolayer films were transferred to
  • Figure 21 summarizes the colorimetric properties and response of the G M1 biosensing monolayer films studied in these experiments showing the initial absorbance, transfer rate, and colorimetric response in buffer and in response to analyte.
  • the initial absorbance (A, mt ) which reflects the maximal peak value of the films at 640 nm, is a function of the film transfer rate and composition.
  • G M consult which does not provide chromatic functionality into the mixed assembly, generally decreases the intensity of the initial blue color.
  • the transfer rate which is the ratio of the area decreased on the tough surface and the area of the substrate emerged into the subphase, indicates that the PDA films are highly transferable as compared to those of sialic acid-PDA (SA-PDA) and G M , molecules.
  • SA-PDA sialic acid-PDA
  • G M G M
  • the blue to red colorimetric response (CR) shows that monolayer films exhibit low CR in buffer solution except when high content of G MI or SA- PDA is used.
  • the ionic content of the aqueous subphase has significant impact on the properties of Langmuir monolayers.
  • the presence of cationic species strengthens the electrostatic interaction of monolayer with anionic headgroups and consequently stabilized the film
  • Figure 22 shows the isotherms of 5% G M ,/5% SA-PDA/90% PDA as a function of subphase concentration of CdCl 2 . As the concentration of Cd 2+ is increased, the expanded phase shifts systematically toward the low molecular area, indicating that the monolayer is stabilized at high Cd 2* concentration.
  • SA-PDA/90% PDA monolayer was observed. This is possibly due to formation of aggregated domains as a result of different ability to interact with Cd 2+ between sialic acid in SA-PDA and G M , and carboxylic in PDA, or precipitation at high salt concentration.
  • Figure 23 shows the isotherms of 5% G MI /5% SA-PDA/90% PDA at pH 4.5, 5.8, and 9.2.
  • pH 9.2 the film became very expanded as a result of electrostatic repulsion between the adjacent PDA molecules. Compression of such a film to form a monolayer was difficult. Additionally, distinct segments of individual molecules were observed, pointing to an immiscible trend in the mixed monolayer that tends to form segregated domains.
  • Figure 24 displays the temperature effect on the isotherms of 100% PDA. 5%SA-PDA/95% PDA. and 5% G M1 /5% SA-PD A/90% PDA. With decrease in subphase temperature, the surface pressure increased and the isotherm shape changed. Isotherms at low temperature exhibited more and more liquid-solid phase transition features, as indicated by the disappearance of the peak and occurrence of the smooth curve at the transition region. All the ⁇ -A isotherms obtained for the three monolayers display similar characteristics. The major difference between these figures is the position of collapse point, which is a function of film composition.
  • Liposomes were prepared using the probe sonication method and polymerized by UV irradiation (254 nm).
  • the colorimetric response was significantly reduced.
  • the enhanced sensitivity observed with the 5,7-docosadiynoic acid liposomes arises from the positioning of the optical reporter group nearer to the interface (i.e., three methylene units compared to eight).
  • the amount of PDA, dopant, and ligand are varied to create the optimal sensor. Although 0-100% amounts are typically used for testing, optimal systems appear to use 5-15% ligands, 0-95% PDA, and 0- 95% dopant. The percent of each component depends on the system, the needed stability, and the needed sensitivity. Certain embodiments of the present invention may inco ⁇ orate more than one type of dopant into the biopolymeric material.
  • Amino-acid derivatized diacetylene dopants were inco ⁇ orated into colorimetric liposomes.
  • the lipids i.e., the dopants and the diacetylene monomers
  • the organic solvent was blown out by use of N 2 gas, and an appropriate amount of water was added to bring the lipid concentration to approximately 1 mM.
  • Bath sonication was used to break down the white precipitate to form liposomes. Typical sonication times varied from 1 hour to 5 hours, dependent on the type of dopants used.
  • the temperature was carefully raised to approximately 80°C to facilitate the formation of the liposomes. The sonication continued until the solutions became clear.
  • the hot solutions were immediately filtered though a 5 ⁇ M Millipore Millex-SV filter to remove any impurity that may be present in the solution.
  • the obtained solutions were stored at 4°C overnight before use.
  • the final liposomes contained the amino-acid derivatized diacetylene dopant.
  • SA- PDA function of SA- PDA is to provide the metastable state of the films for biomolecular recognition through a stress-induced mechanism (Charych et al., Chem. and Biol. 3: 113 [1996]).
  • a film consisting of 1% G MI /1% SA-PDA/98% PDA was also investigated. The CR turned out to be low and it did not yield a useful colorimetric biosensor. As shown in Figure 21, the optimal colorimetric sensor was determined to be 5% G M ,/5% SA-PDA 90% PDA. Thus, a 5% molar content of the dopant SA-PDA provides the best sensor for detection of cholera toxin.
  • Hydrophobic amino acids linked to diacetylenes can be used to lower the solubility of the biopolymeric material as well as the stability of the films or liposomes.
  • These derivatized PDA's can be useful in the assembly of complex systems to fine tune the stability and sensitivity, two factors that are directly coupled to one another.
  • the hydrophobic PDA's with the hydrophilic PDA's the stability of films and liposomes can be greatly increased, under a variety of environmental conditions. Although a large gain in stability is seen, it is at a cost to sensitivity. A balance between sensitivity and stability has to be optimized.
  • Acidic and basic amino acids linked to diacetylenes can be used to increase the solubility of the material. Specifically, these changes allowed polydiacetylene lipids to mix with water soluble biological molecules. Ordinarily, PDA is not water soluble and organic solvents are necessary (i.e., which can be destructive to biological molecules). By placing acidic or basic head groups onto the PDA molecule, the solubility of the derivatized PDA's were greatly enhanced. They also produced much brighter colors and were more consistent in the assembly of sensors. These results were likely due to the increase in water solubility and homogeneity of mixing between all components. The acid/base PDA's were by far the most sensitive of the amino acid-derived diacetylenes.
  • colorimetric biosensors can be made with the addition of fluorescent properties. This provides a multi-pu ⁇ ose and more sensitive sensor.
  • Ligands can be covalently linked to the head groups of self-assembling monomers
  • sialic acid linked to diacetylene monomers can be covalently linked to the surface of polymerized materials (e.g., proteins and antibodies with multiple amine and thiol linkages to the material surface), or can be non-covalently inco ⁇ orated into the biopolymeric material (e.g., ganglioside inco ⁇ orated into the membrane of films and liposomes).
  • the self-assembling monomers can be synthesized to contain a large variety of chemical head-group functionalities using synthesis techniques common in the art.
  • the ligands are then joined to the self-assembling monomers through chemical reaction with these functionalities using synthesis methods well known in the art.
  • the functionalities include, but are not limited to, esters, ethers, amino, amides, thiols, or combinations thereof. Alternately, many ligands can be inco ⁇ orated into the self-assembling matrix without covalent linkage to the surfactants (e.g., membrane proteins and molecules with hydrophobic regions such as gangliosides and lipoproteins).
  • surfactants e.g., membrane proteins and molecules with hydrophobic regions such as gangliosides and lipoproteins.
  • I Sialic Acid Sialic acid was attached as a ligand to diacetylene monomers.
  • Several synthesis methods well known in the art can be used, many of which have general applicability to the attachment of carbohydrates to the inventive biopolymeric materials.
  • PDA 1.0 g, 2.7 mmol in chloroform
  • NHS N-hydroxy succinimide
  • EDC l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the solution was stirred for 2 hours followed by evaporation of the chloroform.
  • Ethyl-5-N-acetyl-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-2-C-(acetic acid)-D-erythro-L- manno-nonate (0.194 g, 0.35 mmol) was added to a cooled solution (5 °C) NHS (0.058 g, 0.50 mmol) and EDC (0.096 g, 0.50 mmol) in 2 ml of chloroform, under nitrogen. The reaction was warmed to ambient temperature with stirring for 5 hours.
  • reaction was then diluted with 15 ml of chloroform and washed with 1 N HC1 (aq.), twice; saturated (aq.) sodium bicarbonate, twice; and saturated (aq.) sodium chloride, once.
  • the organic layer was dried over MgS0 4 , filtered, and evaporated to form ethyl-5-N-acetyl-4,7,8,9-tetra-0-acetyl- 3,5-dideoxy-2-C-(N-succinimidylacetate)-D-erythro-L-manno-nononate.
  • the solution was diluted with 15 ml of chloroform and washed with sodium chloride saturated IN HC1 (aq.), twice; saturated (aq.) sodium bicarbonate, twice; and saturated (aq.) sodium chloride, once.
  • the organic layer was dried over MgS0 4 . filtered, and evaporated to a crude semi-solid.
  • the material was flash chromatographed over silica (20:1 CHCl 3 :MeOH), producing ethyl-5-N-acetyl-4,5,8,9-tetra-0-acetyl-3,5-dideoxy-2-C-[(N-l 1 '- (PDA)-3',6',9'-trioxyundecanyl) acedamido]-D-erythro-L-manno-nononate.
  • the sialic acid derived-PDA was formed by dissolving ethyl-5-N-acetyl-4,5,8,9-tetra- O-acetyl-3,5-dideoxy-2-C-[(N-l r-(PDA)-3',6',9'-trioxyundecanyl) acedamido]-D-erythro-L- manno-nononate (0.20 g, 0.19 mmol) in a solution of 4 ml of water and 0.5 ml of methanol containing 0.1 g dissolved sodium hydroxide.
  • carbohydrates i.e., including sialic acid
  • N-allyl glycosides can then be easily linked to other molecules (e.g., PDA) using simple chemical synthesis methods known in the art.
  • PDA simple chemical synthesis methods known in the art.
  • This method provides a means to inco ⁇ orate a broad range of carbohydrates into biopolymeric material (and thus provides a means to detect a broad range of analytes).
  • oligosaccharides are dissolved in neat allyl amine (water may be added if necessary and does not adversely affect the yield) producing a 0.5-0.1 M solution. The reaction is stopped and stirred for at least 48 hours.
  • the solvent is removed by evaporation and the crude solid is treated with toluene and evaporated to dryness several times.
  • the solid is then chilled in an ice bath and a solution of 60% pyridine, 40% acetic anhydride is added to give a solution containing five hundred mole percent excess of acetic anhydride.
  • the reaction is protected from moisture, stirred and allowed to warm to ambient temperature overnight.
  • the solvents are removed by evaporation and the residue is dissolved in toluene and dried by evaporation several times.
  • the crude product is purified by flash chromatography producing the peracetylated NAc-allyl glycoside form of the free sugars.
  • the peracetylated NAc-allyl glycosides are then dissolved in anhydrous methanol to give a 0.1-0.01 M solution.
  • Several drops of 1 N NaOMe in MeOH are added and the reaction stirred at ambient temperature for 3 hours.
  • Enough Dowex 50 resin (H+ form) is added to neutralize the base, then the solution is filtered and evaporated to dryness (purification by recrystallization can be conducted if desired).
  • the products are the N-allyl glycoslamide form of the carbohydrates.
  • N-allyl glycoslamide forms of a variety of carbohydrates including, but not limited to, glucose, NAc- glucosamine, fucose, lactose, tri-NAc-Chitotriose, Sulfo Lewis x analog, and Sialyl Lewis x analog.
  • Skilled artisans will appreciate the general applicability of this method to the attachment of a broad range of carbohydrates to diacetylene lipids.
  • Ganglioside G M1 Ganglioside G MI presents an example of inco ⁇ oration of a ligand without covalent attachment to the self-assembling monomers.
  • Ganglioside G M was introduced in the biopolymeric material by combining a solution of methanol dissolved ganglioside G M1 (Sigma) with chloroform dissolved PDA, and dried.
  • the ganglioside contains a hydrophobic region that facilitates its inco ⁇ oration into self-assembling surfactant structures.
  • the dried solutions were resuspended in deionized water, the resulting structures contained a mixture of ganglioside and PDA. Liposomes and other forms were produced from the resuspended mixture as described in Example 1.
  • the ganglioside does not contain a polymerizable group, the ganglioside became embedded in the polymerized matrix created by the cross-linking of the diacetylenes. Similar methods can be used for the inco ⁇ oration of other ligands that contain hydrophobic regions (e.g., transmembrane proteins and lipoproteins).
  • the NHS-PDA as generated above, thiol-linked PDA, and other methods known in the art provide functional groups for the attachment of proteins and antibodies.
  • the NHS or thiol-linked monomers are incorporated into the desired aggregate and polymerized.
  • the NHS or thiol functional groups then provide a surface reaction site for covalent linkage to proteins and antibodies using chemical synthesis reactions standard in the art.
  • a hydrazide functional group can be placed on PDA, allowing linkage to aldehydes and ketone groups of proteins and antibodies.
  • NHS-PDA lipid was synthesized as described above. In brief, 1.00 g 10,12- pentacosadiynoic acid (Farchan, Gainesville, FL) was dissolved in CHC1 3 , to which 0.345 g
  • N-hydroxysuccinimide (NHS) and 0.596 g l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added.
  • the solution was stirred at room temperature for two hours, followed by removal of CHC1 3 using a rotavap.
  • the residue was extracted with EtOAC and water. After separation, the organic layer was dried with MgS0 4 and filtered, followed by solvent removal.
  • the raw product was then recrystallized twice with CHC1 3 , and confirmed by FT-IR.
  • the 1 : 1 (molar ratio) PDA/NHS-PDA chloroform solution was spread on the aqueous subphase on a Langmuir-Blodgett trough (KSV mini-trough, KSV Instruments, Inc., Finland) by using a microsyringe (subphase temperature was maintained at 5 °C).
  • the organic solvent was allowed to evaporate by resting the solution for 20 min.
  • the films were compressed to compact monolayer level and then transferred by vertical deposition to glass slides coated with octadecyltrichlorosilane (OTS). The compression and dipping speed was maintained at 5 mm/min. Three layers were deposited onto the glass slide to provide enough colorimetric signal for detection after polymerization and to ensure the hydrophilic surface was exposed to solution.
  • the preparation of stable PDA monolayer films before enzyme immobilization is critical for low background and enhanced reproducibililty of the sensors.
  • the Langmuir monolayer trough provides a method to measure film stability through the evaluation of the surface collapse pressure of the monolayers. It was found that the mixed films (i.e., films with PDA and NHS-PDA) appear to be much more stable than the monolayers consisting of one component and thus more suitable for enzyme immobilization. For instance, the collapse pressure for 1 :1 NHS-PDA/PDA monolayer at 5 °C was 57 mN/m, while NHS-PDA and PDA monolayers collapsed at 34 and 28 mN/m, respectively.
  • the monolayers should possess desirable optical properties (i.e., high color intensity) to be suitable as sensors.
  • Film quality in this particular case color intensity, was studied at different deposition pressures. It was found that films made at 40 mN/m gave the best transfer rate and color intensity. Therefore, the 1 :1 NHS- PDA/PDA films obtained at this transfer pressure were selected for modification with hexokinase.
  • Yeast hexokinase suspension (E.C. 2.7.1.1, from Boehringer Mannheim GmbH, Germany) was spun in a microcentrifuge to remove saturated ammonium sulfate. The protein was resolubilized in 0.1 M phosphate buffer (pH 8.0) to give approximately 1 mg/ml concentration, and dialyzed against the same buffer using a Slide-A-Lyzer dialysis cassette (Pierce) for 3 hours. The PDA monolayer slides were cut into 0.7 cm x 2.5 cm rectangular pieces, and incubated in the hexokinase solution at 4°C for 1 hr.
  • phosphate buffer pH 8.0
  • Antibodies can also be attached to biopolymeric material by hydrazides. In some embodiments, this may be preferred to NHS-coupling because NHS may react at the Fab' region of the antibody, blocking binding to analytes.
  • the hydrazide method causes attachment of the Fc region of the antibodies to the biopolymeric material, leaving available, the binding region. In the hydrazide method, hydrazide-PDA lipids were produced, and unpolymerized liposomes are generated (e.g., 20% hydrazide PDA/80% TRCDA).
  • Uncoupled antibodies are removed from me liposomes by using Centricon 500 filters and washing with 900 ⁇ l Tris buffer (pH 9.0) and centrifugation at 4000 ⁇ m for 2 minutes. After multiple washes, the sample is dilute (if necessary) with Tris buffer to make a 0.2 mM (or less) liposome solution.
  • various other surfactant-linked ligands can be prepared using condensation reactions involving an activated carboxylic acid group and a nucleophilic amino or hydroxy.
  • PDA can be activated with trimethylacetylchloride under anhydrous conditions to form an active asymmetric anhydride.
  • the anhydride can be treated with excess ethylene diamine or ethanolamine to form ethylenediamino-PDA (EDA-PDA) or ethanolamine-PDA
  • EA-PDA EDA-PDA
  • One and a half mole equivalents of triethylamine are added as a catalytic base and reactions are allowed to proceed for three hours at room temperature.
  • EDA-PDA and EA-PDA are chromatographically purified using a silica gel column and a chloroform/methanol gradient.
  • the EDA-PDA or EA-PDA are then be condensed with free carboxylic acid containing ligands (chemically activated as above) to form the ligand-linked polymerizable surfactants.
  • Representative examples of ligands that can be prepared by this method include, but are not limited to, carbohydrates, nucleotides, and biotin.
  • the art contains numerous other examples of successful linkage or association of molecules to lipids and membranes.
  • the self-assembling monomers associated with ligands can be of modified chain length or may consist of double or multiple chains. These various combinations of ligands and monomers provide an extremely broad array of biopolymeric materials appropriate for the interaction with a broad range of analytes, with the desired colorimetric response, selectivity, and sensitivity.
  • the colorimetric changes of the biopolymeric materials of the present invention are detected though simple observation by the human eye. Because of the simplicity of the observation, this function can be accomplished by an untrained observer such as an at-home user.
  • Spectroscopy means may be applied to acquire such data.
  • B 0 is defined as the intensity of abso ⁇ tion at 626 nm divided by the sum of the abso ⁇ tion intensities at 536 and 626 nm.
  • B a represents the new ratio of absorbance intensities after incubation with the analyte.
  • the colorimetric response (CR) of a liposome solution is defined as the percentage change in
  • biopolymeric materials taught by the present invention allow for the detection of numerous analytes.
  • Such analytes range from complex biological organisms (e.g., viruses, bacteria, and parasites) to simple, small organic molecules (e.g., alcohols and sugars).
  • complex biological organisms e.g., viruses, bacteria, and parasites
  • simple, small organic molecules e.g., alcohols and sugars.
  • Specific applications of the presently claimed invention are described below to illustrate the broad applicability of the invention to a range of analyte detection systems and to demonstrate its specificity, and ease of use. These examples are intended to merely illustrate the broad applicability of the present invention. It is not intended that the present invention be limited to these particular embodiments.
  • the presently claimed invention provides superior means of detecting influenza compared to currently available technology. Immunological assays are limited because of me antigenic shift and drift exhibited by the virus. The presently claimed invention detects all varieties of influenza and thus a determination of a patient's exposure to influenza will be definitive, and not limited to a particular strain. Indeed, even newly evolved, uncharacterized influenza strains can be detected.
  • Sialic acid-linked biopolymeric material was generated as described in Examples 1 and 5. The materials were exposed to influenza virus and colorimetric information was observed visually or with spectroscopy as described in Example 6, and shown in Figure 27 for blue (solid line) and red phase (dashed line) material, respectively.
  • a 1-10% mixture of sialic acid-linked PCA was inco ⁇ orated, as previous studies indicated that optimum viral binding occurs for mixtures of 1-10% in liposomes (Spevak et al, J. Am. Chem. Soc. 161: 1146 [1993]).
  • influenza virus detection system include additional ligands that recognize and differentiate influenza strains or serotypes from one another and from other pathogens.
  • sialic-acid containing biopolymeric materials of the present invention provide means of detecting many other pathogens, in addition to influenza virus, sialic acid has the capability of detecting other analytes including, but not limited to, HIV, chlamydia, reovirus,
  • Streptococcus suis Salmonella, Sendai virus, mumps, newcastle, myxovirus, and Neisseria meningitidis.
  • Cholera toxin is an endotoxin of the Gram-negative bacterium Vibrio cholerae that causes potentially lethal diarrheal disease in man. Cholera toxin is composed of two subunits: A (27 kDa) and B (11.6 kDa) with the stoichiometry AB The B components bind specifically to G M , gangliosides on cell surfaces, ultimately leading to translocation of the A, fragment through the membrane.
  • Cholera toxin can be recognized by G M1 -containing supported lipid membranes and polymerized Langmuir-Blodgett films containing G MI and a carbohydrate "promoter" lipid (i.e., sialic acid-derived diacetylenes) as shown by Pan and Charych (Langmuir 13: 1365 [1997]).
  • Ganglioside G M cholera toxin from Vibrio Cholerae, human serum albumin, and wheat germ agglutinin were purchased from Sigma. 5,7-docosadiynoic acid was synthesized. Deionized water was obtained by passing distilled water through a Millipore ⁇ F ultrapurification train. Solvents used were reagent grade. The ganglioside G M1 was mixed at 5 mol % with the diacetylene "matrix lipid" monomers. Liposomes were prepared using the probe sonication method and polymerized by UV irradiation (254 nm).
  • the conjugated ene- yne backbone of polydiacetylene liposomes results in the appearance of a deep blue/pu ⁇ le solution.
  • the visible abso ⁇ tion spectrum of the freshly prepared pu ⁇ le liposomes is shown in Figure 25.
  • cholera toxin was diluted to 1 mg/ml in 50 mM Tris buffer, pH 7.0.
  • blue phase liposomes produced as above were diluted 1 :5 in 50 mM Tris buffer, pH 7.0.
  • the liposomes were pre-incubated in the buffer for 15-30 minutes to ensure stability of the blue phase prior to the addition of cholera toxin. No color changes were observed during this period.
  • Cholera toxin was added to the cuvette by the method of successive additions. After each addition, the contents were mixed and the visible abso ⁇ tion spectrum was recorded as a function of time. Typically, 95% of the absorption changes were observed to occur within the first 2 minutes after addition of toxin as shown in Figure 26. After each experiment, the contents of the cuvette were transferred to a single well of a white microtiter plate. The pink-orange color of the cholera-treated liposomes was verified visually with a blue negative control.
  • Liposomes were prepared with 5% by mole of G M] and 95% 5,7-DCDA.
  • E.coli toxin Sigma
  • the protein was re-diluted in 50 mM Tris buffer pH 7.0 to a final concentration of 1 mg/ml.
  • Figure 29 shows the visible abso ⁇ tion spectrum of the polymeric liposomes containing 5% G M1 ligand and 95% 5,7-DCDA prior to exposure to E. Coli toxin.
  • the liposomes were diluted in 50 mM Tris buffer, pH 8.0 to a final concentration of 0.2 mM in a plastic disposable cuvette.
  • the solution in the cuvette appeared pu ⁇ le to the naked eye.
  • 40 ⁇ l of the above E. coli toxin was added and the sample allowed to incubate for 10 minutes.
  • the visible abso ⁇ tion spectrum was again recorded as shown in Figure 30.
  • the solution in the cuvette appeared pink to the naked eye after the addition of the toxin compared to a pu ⁇ le color before the addition.
  • the absorption spectra of Figures 29 and 30 confirm the color changes observed.
  • the present invention may also be used to detect a variety of other pathogens.
  • Ligands specific for a large number of pathogens (e.g., carbohydrates, proteins, and antibodies) can be inco ⁇ orated into the biopolymeric material using routine chemical synthesis methods described above and known in the art.
  • Some of the examples of pathogen detection systems are presented below to demonstrate the variety of methods that can be applied using the present invention and to demonstrate the broad detecting capabilities of single ligand species (e.g., sialic acid).
  • the sialic acid derivated material of the present invention has been used to detect the presence of parasites such as Plasmodium (i. e. , the etiologic agent that causes malaria).
  • the genetically conserved host binding site was utilized.
  • PDA films containing sialic acid as described above were exposed to solutions containing malaria parasites and erythrocytes. After overnight exposure to the parasites, the films became pink in color. The color response (CR) in each case was nearly 100%.
  • the system be used in conjunction with other testing material (e.g., arrays of biopolymeric material with various ligands) to identify and differentiate the presence of particularly virulent species or strains of Plasmodium (e.g., P. falciparum) or other pathogens.
  • antibodies were used as ligands to successfully detect the presence of Neisseria gonorrhoeae and Vibrio vulnificus.
  • the inco ⁇ oration of the antibodies into the biopolymeric material is described in Example 5.
  • the present invention provides a variety of means to detect a broad range of pathogens, including bacteria, viruses, and parasites.
  • V. Detection of Volatile Organic Chemicals Certain embodiments of the presently claimed invention provide means to colorimetrically detect volatile organic compounds (VOCs). Most of the current methods of VOC detection require that samples be taken to laboratory facilities where they are analyzed by gas chromatography/mass spectroscopy. Some of the on-site methodologies require large, bulky pieces of equipment such as that used in spectroscopic analysis. While these methods are excellent for providing quantitation and identification of the contaminant, they cannot ensure the safety of the individual worker. In one embodiment, the present invention provides a badge containing immobilized biopolymeric material that signals the presence of harmful VOCs and provides maximum workplace safety within areas that contain VOCs.
  • the badge is easy and simple to read and requires no expertise to analyze on the part of the wearer.
  • the color change of the badge signals the individual to take appropriate action.
  • the badges reduce costs and improve the efficiency of environmental management and restoration actions, significantly reducing down-time due to worker illness by preventing over-exposure to potentially harmful substances.
  • the quartz crystal microbalance (QCM) and the surface acoustic wave (SAW) devices have linear frequency changes with applied mass.
  • QCM quartz crystal microbalance
  • SAW surface acoustic wave
  • a sensor based on the QCM or SAW is constructed.
  • the complex electronics involved in the use of SAW, QCM, and electrode based systems makes these approaches less amenable to use as personal safety devices.
  • the present invention differs from these methods in that signal transduction is an integral part of the organic layer structure rather than signal transduction to an electronic device.
  • embodiments of the present invention facilitate optical detection of the signal rather than electronic detection.
  • the present invention provides flexibility in material design, allowing easy immobilization into a small cartridge (e.g., a badge) rather than being burdened with the need for electronic equipment.
  • curve a shows the abso ⁇ tion spectrum of a PCA film in blue phase.
  • the film changes to red phase PCA, curve b, upon exposure to approximately 500 ppm of 1-octanol dissolved in water.
  • the degree of color change was generally dependent upon the concentration of the solvent and also increased with the extent of halogenation and aromaticity.
  • a single component thin membrane film of PCA was prepared and polymerized to the blue state by UV exposure (254 nm).
  • the y-axis represents the colorimetric response, or the extent of blue-to-red conversion.
  • the numbers above the bar represent an upper limit to the detection in ppm. For many of these solvents, it is clear that solvent concentrations well below 500 ppm can be detected.
  • the pharmaceutical industry has an ongoing need for solvent sensors, as pharmaceutical compounds are typically manufactured through organic chemical reactions that take place in the presence of solvents.
  • the solvent Before packaging of a drug for use in humans or other animals, the solvent must be completely driven off (Carey and Kowalski, Anal. Chem. 60: 541 [1988]).
  • the currently used method for detecting these VOCs uses energy intensive dryers to blow hot air across the drug and piezoelectric crystal arrays to analyze the evaporation of the various solvents (Carey, Trends in Anal. Chem. 13: 210 [1993]).
  • the presently claimed invention provides a colorimetric based approach that greatly simplify these measurements.
  • compound 1 has a pronounced affinity for dioxane and little affinity for butanol, acetone, methanol, 2-propanol, cyclohexane, toluene, and water.
  • Compound 2 shows a pronounced affinity for 1 -butanol over the same group of solvents.
  • the pu ⁇ ose of this example is to show the development of a new class of functional materials that specifically trap small organic compounds and report the entrapment event by a colorimetric change which can be detected visually. These material act as simple color-based sensor devices that detects the presence of compounds such as solvents or other toxic pollutants in air or water streams.
  • the first step involves the synthesis of lipid diacetylene analogs of compounds 1 and 2 as shown in Figure 34.
  • the enantiometrically pure ester of PDA (pentacosadiynoic acid) 3 is hydroxylated via molybdenum peroxide oxidation to alcohol 4.
  • Diasteriomers are separated and the ester is hydrolyzed to chiral lactate analogs 5 and 6.
  • the ethyl esters are formed and treated with Grignard reagents to give the desired chiral lipid analogs 7 and 8.
  • Variation in the R groups result in a wide variety of new materials in which the specific entrapment capabilities are reviewed.
  • the monomer-lipid clathrate is ordered and compressed on the water surface using a
  • the hexokinase modified films were placed onto silanized glass cover slides for the pu ⁇ ose of measuring the optical properties.
  • the biosensor coated glass cover slides were placed in glass cuvettes and the UV- Vis spectra of hexokinase modified films were recorded in 0.1 M phosphate buffer (pH 6.5).
  • analytes detectable by the presently claimed invention ranging from complex biological organisms (e.g., viruses, bacteria, and parasites) to simple, small organic molecules (e.g., alcohols).
  • complex biological organisms e.g., viruses, bacteria, and parasites
  • simple, small organic molecules e.g., alcohols.
  • ligands linked to biopolymeric material including, but not limited to botulinum neurotoxin detected with ganglioside inco ⁇ orated PDA (Pan and Charych, Langmuir 13: 1367 [1997]). It is contemplated that numerous ligand types will be linked to self-assembling monomers using standard chemical synthesis techniques known in the art to detect a broad range of analytes.
  • ligand types can be inco ⁇ orated into the biopolymeric matrix without covalent attachment to self-assembling monomer. These materials ailow for the detection of small molecules, pathogens, bacteria, membrane receptors, membrane fragments, volatile organic compounds, enzymes, drugs, and many other relevant materials.
  • the presently claimed invention also finds use as a sensor in a variety of other applications.
  • the color transition of PDA materials is affected by changes in temperature and pH.
  • the methods and compositions of the presently claimed invention find use as temperature and pH detectors.
  • Ligands can also be used in the present invention when they function as competitive binders to the analyte. For example, by measuring the colorimetric response to an analyte in the presence of a natural receptor for the analyte, one can determine the quantity and/or binding affinity of the natural receptor.
  • Competition or inhibition techniques allow the testing of very small, largely unreactive compounds, as well as substances present in very low concentrations or substances that have a small number or single valiancy.
  • One application of this technique finds use as a means for the development and improvement of drugs by providing a screening assay to observe competitive inhibition of natural binding events.
  • the compositions of the presently claimed invention further provide means for testing libraries of materials, as the binding of desired material can be colorimetrically observed and the relevant biopolymeric material with its relevant ligand separated from the others by segregating out a particular polymeric structure.
  • the silicon gel or wafers are acid cleaned in 1 :1 HCl/methanol, rinsed in water, and placed in concentrated sulfuric acid. After a thorough water rinse, the wafer chips or gel is boiled in doubly distilled deionized water, allowed to cool and dry and then silanized under inert atmosphere in a 2% solution of 3-mercaptopropyl trimethoxysilane prepared in dry toluene.
  • the chips or gels are placed in a 2 mM solution of either GMBS (N- succinimidyl 4-maleimidobutyrate) or EMCS (N-succinimidyl 6-maleimidocaproate) prepared in 0.1 M phosphate buffer (the cross linker is first dissolved in a minimal amount of dimethylformamide). After rinsing with phosphate buffer, the chips are placed in a 0.05 mg/ml solution of the liposomes prepared in pH 8.0 phosphate buffer. Finally, the chips or gels are thoroughly rinsed with, and then stored in, the buffer solution prior to their use.
  • the liposomes should have an -NH 2 functionality for the cross-linking with GMBS or EMCS to work.
  • a silica sol was prepared by sonicating 15.25 g of tetramethylorthosilicate (TMOS), 3.35 g of water, and 0.22 ml of 0.04 N aqueous hydrochloric acid in a chilled bath until the solution was one phase (approximately 20 minutes). Chilled MOPS buffer solution (50% v/v) was then added to the acidic sol making sure that the solution was well cooled in an ice bath to retard gelation.
  • TMOS tetramethylorthosilicate
  • TMOS tetramethylorthosilicate
  • TEOS tetraethylorthosilicate
  • MeTEOS methyltriethoxysilane
  • aryl silsesquioxanes find use in generating sol-gel glass.
  • a polymerized liposome solution (2.5 ml) (as generated in Example 1) was then mixed into the buffered sol (10 ml) and the mixture poured into plastic cuvettes, applied as a film on a flat surface, or poured into any other desired formation template, sealed with Parafilm, and allowed to gel at ambient temperature. Gelation of the samples occurred within a few minutes resulting in transparent, monolithic solids (18 mm x
  • biopolymeric material shapes . e. , film and other nanostructures
  • the materials must be generated or sectioned into small (i.e., nanoscopic) sized portions if not already so, and inco ⁇ orated into a solution to be mixed with the buffered sol.
  • the presently claimed invention contemplates the generation of a large palette of polymerizable lipids of different headgroup chemistries to create an array.
  • Lipids containing head groups with carboxylic acid functionalities (imparting a formal negative charge), hydrophilic uncharged hydroxy groups, primary amine functionalities (that may acquire a formal positive charge), amino derivatives (with positive, negative or zwitterionic charge), and hydrophobic groups among others can be generated.
  • the combination of these materials into a single device facilitates the simultaneous detection of a variety of analytes or the discrimination of a desired analytes from background interferants.
  • biopolymeric materials comprising varying dopant materials are used to provide a different color pattern for each portion of the array.
  • lipids with various head group chemistries can be generated to create an array.
  • Figure 37 depicts lipids with various head group chemistries. These may be categorized into five groups based upon their head group functionality.
  • Compounds 2.4 and 2.5 contain carboxylic acid functionalities, imparting a formal negative charge.
  • Compounds 2.6 and 2.7 contain a hydrophilic uncharged hydroxyl group.
  • Compounds 2.8 and 2.9 have primary amine functionalities that may acquire a formal positive charge.
  • the amino acid derivative 2.10 may exist with positive, negative or zwitterionic charge.
  • Compounds 2.11-2.13 have hydrophobic head groups.
  • the synthesis of these lipids begins with commercially available PDA (2.4). Synthesis of all but 2.10, 2.12, and 2.13 can be carried out by coupling the respective head group to PDA utilizing the activated N-hyroxysuccinimidyl ester of PDA (NHS-PDA) as described above.
  • the amino acid lipid 2.10 can be prepared in four steps from PDA as shown in Figure 38. using lithium aluminum hydride and transformation of the alcohol to the corresponding bromide derivative. The bromide is converted to the protected amino acid by reaction with diethyl N-acetimidomalonate in acetonitrile with sodium hydride, followed by deprotection.
  • the fluorinated lipids 2.12 and 2.13 can be prepared by the reaction of pentafluorobenzoyl chloride with amino lipids 2.8 and 2.9.
  • Materials prepared as above can be deposited into chambers of a device or immobilized to specific portions of a device.
  • a single apparatus e.g., a badge
  • an array is generated with the ability to identify, distinguish, and quantitate a broad range of reactions and analytes.
  • Biopolymeric liposomes were prepared by probe sonication of a mixture of polymerizable matrix lipid 10,12-tricosadiynoic acid and various mole fractions (0%-40%) of PLA 2 substrate lipid (e.g., DMPC) in water, followed by polymerization with 1.6 ⁇ J/cm 2 ultraviolet radiation, 254 nm. Analysis by transmission electron microscopy indicated an average vesicle size of approximately 100 nm.
  • PLA 2 substrate lipid e.g., DMPC
  • Liposomes containing a range of mole% DMPC were tested for their ability to produce a colorimetric response. Five microliters of 1.4 mg/ml PLA 2 was added to 50 ⁇ l of
  • DMPC/PDA vesicles (0.1 mM final total lipid concentration).
  • the experiment was carried out in a standard 96-well plate using a Molecular Devices UV Max kinetic microplate reader. The absorption of the vesicle solution was monitored as a function of time at 620 nm and 490 nm wavelengths. The data was then plotted as colorimetric response (CR) versus time to yield the color response curves as shown in Figure 17, described above.
  • CR colorimetric response
  • PLA 2 activity was independently measured using a labeled lipid analog inco ⁇ orated into the PDA matrix, allowing simultaneous measurement of product formation and colorimetric response of the vesicles.
  • the analog used was thioester l,2-bis-(S-decanoyl)-l,2-dithio-sn-glycero-3- phosphocholine (DTPC).
  • DTPC thioester l,2-bis-(S-decanoyl)-l,2-dithio-sn-glycero-3- phosphocholine
  • Five microliters of 40% DTPC/PDA vesicles diluted with 45 ⁇ l 40 mM Tris pH 7.0 and 5 ⁇ l of 6 mM DTNB were incubated with 10 ⁇ l of 1.4 mg/ml PLA 2 .
  • the absorbance at 412 nm was monitored over time.
  • NMR experiments were conducted to further verify the occurrence of interfacial catalysis by PLA 2 , and provide information of the fate of the enzymatic reaction products.
  • the spectra were taken at a magnetic field of 11.7 Tesla on a Bruker DMX500 NMR spectrometer.
  • the Block-decay pulse sequence was used with 2048 acquisition data points. 40 000 free induction decays were accumulated in each experiment with 2 second recycle delays. 0.1 M phosphoric acid was used as an external reference.
  • Figure 16 shows the 31 P NMR spectra of A) Mixed DMPC/PDA vesicles, 0.1 mM total lipid; B) the same vesicle suspension after addition of PLA 2 (200 ng).
  • the assays for phospholipase D and C were run under similar conditions as the phospholipase PLA 2 assays. In all assays, 1 mM 40% DMPC/ 60% 10,12-tricosadiynoic acid (TRCDA) liposomes were used. Aqueous stock solutions of phospholipase D and C were prepared by dissolving the enzymes at 1 mg/ml concentration in 50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 pH 8.9 buffer and 20 mM sodium borate, 150 mM NaCl, 5 mM CaCl 2 pH 8.9 buffer, respectively.
  • TRCDA 10,12-tricosadiynoic acid
  • the assays were then performed by adding 5 ⁇ l of liposomes, 45 ⁇ l 50 mM Tris pH 7.0 (or 20 mM sodium borate pH 7.0 when testing PLC), and 5 ⁇ l of enzyme. Controls for the assays consisted of 5 ⁇ l of buffer instead of enzyme. The assays were monitored at 620 nm and 490 nm every two minutes for the first ten minutes, and then every ten minutes for the remaining 50 minutes.
  • Inhibitor Screening Inhibitors were used to block the colorimetric event initiated by PLA 2 .
  • DMPC/PDA vesicles containing 0.6% MJ33 were polymerized and incubated with 5 ⁇ l of 1.4 mg/ml PLA 2 .
  • Five microliters of unpolymerized liposomes were combined with 40 ⁇ l of 50 mM Tris pH 7.0, 5 ⁇ l MJ33 (0.006 M dissolved in water), 5 ⁇ l of 50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 pH 8.9, and incubated for 15 minutes.
  • the liposomes were then polymerized in 96 well plates and abso ⁇ tion spectrum were recorded at 490 nm and 620 nm. Five microliters of PLA 2 were added and spectra at specific time intervals were monitored for one hour. For Zn 2+ inhibition, the enzyme was dissolved in 10 mM Tris. 150 mM NaCl, 0.1 mM ZnCl 2 pH 8.9.
  • PCT/US98/03963 (81) Designated States: AU, CA, JP, European patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
  • the present invention relates to methods and compositions for the direct detection of membrane conformational changes through the detection of color changes in biopolymeric materials.
  • the present invention allows for the direct colorimetric detection of membrane modifying reactions and analytes responsible for such modifications and for the screening of reaction inhibitors.
  • a method for detecting a reaction comprising: a) providing: i) biopolymeric material comprising reaction substrate and a plurality of self-assembling monomers; and ii) a reaction means; b) exposing said reaction means to said biopolymeric material; and c) detecting a color change in said biopolymeric material which indicates at least a partial occurrence of said reaction.
  • reaction means comprises a lipid cleavage means.
  • biopolymeric materials are selected from the group consisting of liposomes, films, tubules, helical assemblies, fiber-like assemblies, and solvated polymers.
  • said self assembling monomers comprise diacetylene monomers selected from the group consisting of 5,7-docosadiynoic acid, 5,7- pentacosadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof.
  • said self-assembling monomers are selected from the group consisting of acetylenes, alkenes, thiophenes, polythiophenes, siloxanes, poly- silanes, anilines, pyrroles, polyacetylenes, poly (para-phylenevinylene), poly (para-phylene), vinylpyridinium, and combinations thereof.
  • said biopolymeric material further comprises one or more ligands.
  • biopolymeric material further comprises one or more dopants.
  • said one or more dopants is selected from the group consisting of surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, steroids, cerebroside, lysophosphatidylcholine, D- erythroshingosine, sphingomyelin, dodecyl phosphocholine, N-biotinyl phosphatidylethanolamine and combinations thereof.
  • surfactants polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol
  • said one or more dopants comprises diacetylene derivatives selected from the group consisting of sialic acid-derived diacetylene, lactose-derived diacetylene, amino acid-derived diacetylene, and combinations thereof.
  • biopolymeric material further comprises a support, and wherein said biopolymeric material is immobilized to said support.
  • said support is selected from the group consisting of polystyrene, polyethylene, teflon, mica, sephadex, sepharose, polyacrynitriles, filters, glass, gold, silicon chips, and silica.
  • a method for detecting the presence of an analyte comprising providing biopolymeric material comprising analyte substrate and a plurality of self-assembling monomers: exposing a sample suspected of containing said analyte to said biopolymeric material; and detecting a color change in said biopolymeric material, which indicates the presence of said analyte.
  • biopolymeric materials are selected from the group consisting of liposomes, films, tubules, helical assemblies, fiber-like assemblies, and solvated polymers
  • said self-assembling monomers comprise diacetylene monomers selected from the group consisting of 5,7-docosadiynoic acid, 5,7- pentacosadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof.
  • said one or more ligands is selected from the group consisting of proteins, antibodies, carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, short peptides, pepstatin, Diels-Alder reagents, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, trisaccharides, tetrasaccharides, ganglioside G M1 , ganglioside G Tlb , sialic acid, and combinations thereof.
  • said one or more dopants is selected from the group consisting of surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, steroids, cerebroside, lysophosphatidylcholine, D- erythroshingosine, sphingomyelin, dodecyl phosphocholine, N-biotinyl phosphatidylethanolamine and combinations thereof.
  • surfactants polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol
  • said one or more dopants comprises diacetylene derivatives selected from the group consisting of sialic acid-derived diacetylene, lactose-derived diacetylene, amino acid-derived diacetylene, and combinations thereof.
  • biopolymeric material further comprises a support, and wherein said biopolymeric material is immobilized to said support.
  • said support is selected from the group consisting of polystyrene, polyethylene, teflon, mica, sephadex, sepharose, polyacrynitriles, filters, glass, gold, silicon chips, and silica.
  • a method for detecting inhibitors comprising: a) providing: i) biopolymeric material comprising reaction substrate and a plurality of self-assembling monomers; ii) a reaction means; and iii) a sample suspected of containing an inhibitor; b) combining said biopolymeric material and said sample suspected of containing an inhibitor; c) exposing said biopolymeric material and said sample suspected of containing an inhibitor to said reaction means; and d) detecting the presence or absense of a color change in said biopolymeric material, thereby detecting the activity of said inhibitor.
  • reaction means comprises a cleavage means.
  • biopolymeric materials are selected from the group consisting of liposomes, films, tubules, helical assemblies, fiber-like assemblies, and solvated polymers
  • said self-assembling monomers comprise diacetylene monomers selected from the group consisting of 5,7-docosadiynoic acid, 5,7- pentacosadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof.
  • said one or more ligands is selected from the group consisting of proteins, antibodies, carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, short peptides, pepstatin, Diels-Alder reagents, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, trisaccharides, tetrasaccharides, ganglioside G M consult ganglioside G Tlb , sialic acid, and combinations thereof.
  • biopolymeric material further comprises one or more dopants.
  • said one or more dopants is selected from the group consisting of surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide.
  • said one or more dopants comprise diacetylene derivatives selected from the group consisting of sialic acid-derived diacetylene, lactose-derived diacetylene, amino acid-derived diacetylene, and combinations thereof.
  • biopolymeric material further comprises a support, and wherein said biopolymeric material is immobilized to said support.

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Abstract

La présente invention concerne des procédé et des compositions permettant détection directe de transconformation membranaire par la détection d'évolutions des couleurs dans des matériaux biopolymères. L'invention concerne plus particulièrement, d'une part la détection directe par procédé colorimétriques de réactions de modifications de la membrane et d'analytes responsables de telles modifications, et d'autre part la recherche d'inhibiteurs de la réaction.
EP98907684A 1997-03-03 1998-03-02 Detection directe de biocatalyseurs par procede colorimetrique Withdrawn EP0965033A1 (fr)

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US6080423A (en) 1994-08-11 2000-06-27 Regents Of The University Of California Three dimensional colorimetric assay assemblies
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US6468759B1 (en) 2002-10-22
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AU742885B2 (en) 2002-01-17
JP2002515980A (ja) 2002-05-28
CA2282433A1 (fr) 1998-09-11

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